Publications by authors named "Natasha T Snider"

37 Publications

The elegant complexity of mammalian ecto-5'-nucleotidase (CD73).

Trends Cell Biol 2021 Jun 8. Epub 2021 Jun 8.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Purinergic signaling is a fundamental mechanism used by all cells to control their internal activities and interact with the environment. A key component of the purinergic system, the enzyme ecto-5'-nucleotidase (CD73) catalyzes the last step in the extracellular metabolism of ATP to form adenosine. Efforts to harness the therapeutic potential of endogenous adenosine in cancer have culminated in the ongoing clinical development of multiple CD73-targeting antibodies and small-molecule inhibitors. However, recent studies are painting an increasingly complex picture of CD73 mRNA and protein regulation and function in cellular homeostasis, physiological adaptation, and disease development. This review discusses the latest conceptual and methodological advances that are helping to unravel the complexity of this important enzyme that was identified nearly 90 years ago.
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http://dx.doi.org/10.1016/j.tcb.2021.05.008DOI Listing
June 2021

Assembly of NFL and desmin intermediate filaments: Headed in the right direction.

Proc Natl Acad Sci U S A 2021 Mar;118(13)

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

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http://dx.doi.org/10.1073/pnas.2102176118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020673PMC
March 2021

CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner.

Cell Mol Gastroenterol Hepatol 2021 Jan 29;12(1):141-157. Epub 2021 Jan 29.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

Background & Aims: Metabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. CD73 is the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. We aimed to determine if CD73 has nonredundant hepatoprotective functions.

Methods: Liver-specific CD73 knockout (CD73-LKO) mice were generated by targeting the Nt5e gene in hepatocytes. The CD73-LKO mice and hepatocytes were characterized using multiple approaches.

Results: Deletion of hepatocyte Nt5e resulted in an approximately 70% reduction in total liver CD73 protein (P < .0001). Male and female CD73-LKO mice developed normally during the first 21 weeks without significant liver phenotypes. Between 21 and 42 weeks, the CD73-LKO mice developed spontaneous-onset liver disease, with significant severity in male mice. Middle-aged male CD73-LKO mice showed hepatocyte swelling and ballooning (P < .05), inflammation (P < .01), and variable steatosis. Female CD73-LKO mice had lower serum albumin levels (P < .05) and increased inflammatory genes (P < .01), but did not show the spectrum of histopathologic changes in male mice, potentially owing to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice showed significant metabolic imbalance, with increased blood urea nitrogen (P < .0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypophosphorylation of AMPK substrates in CD73-LKO livers (P < .0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (P < .001).

Conclusions: Hepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.
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http://dx.doi.org/10.1016/j.jcmgh.2021.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082562PMC
January 2021

Alternative Splicing in Hepatocellular Carcinoma.

Cell Mol Gastroenterol Hepatol 2020 8;10(4):699-712. Epub 2020 May 8.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases, with more than 850,000 new diagnoses per year globally. Recent trends in the United States have shown that liver cancer mortality has continued to increase in both men and women, while 5-year survival remains below 20%. Understanding key mechanisms that drive chronic liver disease progression to HCC can reveal new therapeutic targets and biomarkers for early detection of HCC. In that regard, many studies have underscored the importance of alternative splicing as a source of novel HCC prognostic markers and disease targets. Alternative splicing of pre-mRNA provides functional diversity to the genome, and endows cells with the ability to rapidly remodel the proteome. Genes that control fundamental processes, such as metabolism, cell proliferation, and apoptosis, are altered globally in HCC by alternative splicing. This review highlights the major splicing factors, RNA binding proteins, transcriptional targets, and signaling pathways that are of key relevance to HCC. We highlight primary research from the past 3-5 years involving functional interrogation of alternative splicing in rodent and human liver, using both large-scale transcriptomic and focused mechanistic approaches. Because this is a rapidly advancing field, we anticipate that it will be transformative for the future of basic liver biology, as well as HCC diagnosis and management.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490524PMC
May 2020

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

Hepatol Commun 2019 Oct 9;3(10):1400-1414. Epub 2019 Aug 9.

Department of Cell Biology and Physiology University of North Carolina at Chapel Hill Chapel Hill NC.

CD73, a cell-surface -linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other -linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal -linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity ( 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity , similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that -linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.
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http://dx.doi.org/10.1002/hep4.1410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771166PMC
October 2019

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Am J Physiol Cell Physiol 2019 12 28;317(6):C1079-C1092. Epub 2019 Aug 28.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.
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http://dx.doi.org/10.1152/ajpcell.00285.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957383PMC
December 2019

Vimentin on the move: new developments in cell migration.

F1000Res 2018 15;7. Epub 2018 Nov 15.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC, USA.

The vimentin gene ( ) encodes one of the 71 human intermediate filament (IF) proteins, which are the building blocks of highly ordered, dynamic, and cell type-specific fiber networks. Vimentin is a multi-functional 466 amino acid protein with a high degree of evolutionary conservation among vertebrates. mice, though viable, exhibit systemic defects related to development and wound repair, which may have implications for understanding human disease pathogenesis. Vimentin IFs are required for the plasticity of mesenchymal cells under normal physiological conditions and for the migration of cancer cells that have undergone epithelial-mesenchymal transition. Although it was observed years ago that vimentin promotes cell migration, the molecular mechanisms were not completely understood. Recent advances in microscopic techniques, combined with computational image analysis, have helped illuminate vimentin dynamics and function in migrating cells on a precise scale. This review includes a brief historical account of early studies that unveiled vimentin as a unique component of the cell cytoskeleton followed by an overview of the physiological vimentin functions documented in studies on mice. The primary focus of the discussion is on novel mechanisms related to how vimentin coordinates cell migration. The current hypothesis is that vimentin promotes cell migration by integrating mechanical input from the environment and modulating the dynamics of microtubules and the actomyosin network. These new findings undoubtedly will open up multiple avenues to study the broader function of vimentin and other IF proteins in cell biology and will lead to critical insights into the relevance of different vimentin levels for the invasive behaviors of metastatic cancer cells.
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http://dx.doi.org/10.12688/f1000research.15967.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241562PMC
March 2019

The sweet side of vimentin.

Elife 2018 03 7;7. Epub 2018 Mar 7.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, United States.

A protein modification called O-linked glycosylation regulates the interactions between vimentin molecules under normal conditions, and the ability of bacteria to replicate after they infect cells.
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http://dx.doi.org/10.7554/eLife.35336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841928PMC
March 2018

An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

FASEB J 2018 05 18;32(5):2841-2854. Epub 2018 Jan 18.

Department of Cell Biology and Physiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA.

Vimentin is a cytoskeletal intermediate filament protein that is expressed in mesenchymal cells and cancer cells during the epithelial-mesenchymal transition. The goal of this study was to identify vimentin-targeting small molecules by using the Tocriscreen library of 1120 biochemically active compounds. We monitored vimentin filament reorganization and bundling in adrenal carcinoma SW13 vimentin-positive (SW13-vim) cells via indirect immunofluorescence. The screen identified 18 pharmacologically diverse hits that included 2 statins-simvastatin and mevastatin. Simvastatin induced vimentin reorganization within 15-30 min and significant perinuclear bundling within 60 min (IC = 6.7 nM). Early filament reorganization coincided with increased vimentin solubility. Mevastatin produced similar effects at >1 µM, whereas the structurally related pravastatin and lovastatin did not affect vimentin. In vitro vimentin filament assembly assays revealed a direct targeting mechanism, as determined biochemically and by electron microscopy. In SW13-vim cells, simvastatin, but not pravastatin, reduced total cell numbers (IC = 48.1 nM) and promoted apoptosis after 24 h. In contrast, SW13-vim cell viability was unaffected by simvastatin, unless vimentin was ectopically expressed. Simvastatin similarly targeted vimentin filaments and induced cell death in MDA-MB-231 (vim), but lacked effect in MCF7 (vim) breast cancer cells. In conclusion, this study identified vimentin as a direct molecular target that mediates simvastatin-induced cell death in 2 different cancer cell lines.-Trogden, K. P., Battaglia, R. A., Kabiraj, P., Madden, V. J., Herrmann, H., Snider, N. T. An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.
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http://dx.doi.org/10.1096/fj.201700663RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901388PMC
May 2018

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications.

J Vis Exp 2017 05 18(123). Epub 2017 May 18.

Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill;

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton - a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.
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http://dx.doi.org/10.3791/55655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603907PMC
May 2017

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

PLoS One 2016 11;11(8):e0160982. Epub 2016 Aug 11.

Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI, 48109, United States of America.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals during cellular stress via several post-translational modifications that change its folding properties, protein-protein interactions and sub-cellular localization. We examined GAPDH properties in acute mouse liver injury due to ethanol and/or acetaminophen (APAP) treatment. Synergistic robust and time-dependent nuclear accumulation and aggregation of GAPDH were observed only in combined, but not individual, ethanol/APAP treatments. The small molecule GAPDH-targeting compound TCH346 partially attenuated liver damage possibly via mitochondrial mechanisms, and independent of nuclear accumulation and aggregation of GAPDH. These findings provide a novel potential mechanism for hepatotoxicity caused by combined alcohol and acetaminophen exposure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160982PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981434PMC
August 2017

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Methods Mol Biol 2016 ;1412:227-36

Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as of endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid-metabolizing P450s and their corresponding products, by using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.
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http://dx.doi.org/10.1007/978-1-4939-3539-0_23DOI Listing
December 2017

Kidney keratins: cytoskeletal stress responders with biomarker potential.

Authors:
Natasha T Snider

Kidney Int 2016 Apr;89(4):738-40

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. Electronic address:

Keratins are cytoskeletal filamentous proteins that support the structural integrity of epithelial cells. Deficiency of the major simple epithelial keratins K8, K18, and K19 increases susceptibility to hepatobiliary injury, but keratin function in kidney injury has not been addressed. Djudjaj et al. examined renal keratins in health and disease, in both mice and humans. Their findings lay the foundation for pursuing keratins as markers and regulators of renal tubular epithelial injury.
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http://dx.doi.org/10.1016/j.kint.2015.12.040DOI Listing
April 2016

Assays for Posttranslational Modifications of Intermediate Filament Proteins.

Methods Enzymol 2016 6;568:113-38. Epub 2015 Nov 6.

Department of Molecular & Integrative Physiology, Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA.

Intermediate filament (IF) proteins are known to be regulated by a number of posttranslational modifications (PTMs). Phosphorylation is the best-studied IF PTM, whereas ubiquitination, sumoylation, acetylation, glycosylation, ADP-ribosylation, farnesylation, and transamidation are less understood in functional terms but are known to regulate specific IFs under various contexts. The number and diversity of IF PTMs is certain to grow along with rapid advances in proteomic technologies. Therefore, the need for a greater understanding of the implications of PTMs to the structure, organization, and function of the IF cytoskeleton has become more apparent with the increased availability of data from global profiling studies of normal and diseased specimens. This chapter will provide information on established methods for the isolation and monitoring of IF PTMs along with the key reagents that are necessary to carry out these experiments.
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http://dx.doi.org/10.1016/bs.mie.2015.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855289PMC
October 2016

PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding.

Hepatology 2015 Dec 25;62(6):1858-69. Epub 2015 Aug 25.

Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.

Unlabelled: Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments.

Conclusion: The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases.
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http://dx.doi.org/10.1002/hep.27965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681638PMC
December 2015

Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5'-nucleotidase (CD73).

Mol Biol Cell 2014 Dec 8;25(25):4024-33. Epub 2014 Oct 8.

Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109 Departments of Medicine, University of Michigan, Ann Arbor, MI 48109.

Ecto-5'-nucleotidase (CD73), encoded by NT5E, is the major enzymatic source of extracellular adenosine. CD73 controls numerous pathophysiological responses and is a potential disease target, but its regulation is poorly understood. We examined NT5E regulation by alternative splicing. Genomic database analysis of human transcripts led us to identify NT5E-2, a novel splice variant that was expressed at low abundance in normal human tissues but was significantly up-regulated in cirrhosis and hepatocellular carcinoma (HCC). NT5E-2 encodes a shorter CD73 isoform we named CD73S. The presence of CD73S protein, which lacks 50 amino acids, was detected in HCC using an isoform-specific antibody. A noncanonical mouse mRNA, similar to human CD73S, was observed, but the corresponding protein was undetectable. The two human isoforms exhibited functional differences, such that ectopic expression of canonical CD73 (CD73L) in human HepG2 cells was associated with decreased expression of the proliferation marker Ki67, whereas CD73S expression did not have an effect on Ki67 expression. CD73S was glycosylated, catalytically inactive, unable to dimerize, and complexed intracellularly with the endoplasmic reticulum chaperone calnexin. Furthermore, CD73S complexed with CD73L and promoted proteasome-dependent CD73L degradation. The findings reveal species-specific CD73 regulation, with potential significance to cancer, fibrosis, and other diseases characterized by changes in CD73 expression and function.
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http://dx.doi.org/10.1091/mbc.E14-06-1167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263446PMC
December 2014

Modulation of cytoskeletal dynamics by mammalian nucleoside diphosphate kinase (NDPK) proteins.

Naunyn Schmiedebergs Arch Pharmacol 2015 Feb 20;388(2):189-97. Epub 2014 Sep 20.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA,

Nucleoside diphosphate kinase (NDPK) proteins comprise a family of ten human isoforms that participate in the regulation of multiple cellular processes via enzymatic and nonenzymatic functions. The major enzymatic function of NDPKs is the generation of nucleoside triphosphates, such as guanosine triphosphate (GTP). Mechanisms behind the nonenzymatic NDPK functions are not clear but likely involve context-dependent signaling roles of NDPK within multi-protein complexes. This is most evident for NDPK-A, which is encoded by the human NME1 gene, the first tumor metastasis suppressor gene to be identified. Understanding which protein interactions are most relevant for the biological and metastasis-related functions of NDPK will be important in the potential utilization of NDPK as a disease target. Accumulating evidence suggests that NDPK interacts with and affects various components and regulators of the cytoskeleton, including actin-binding proteins, intermediate filaments, and cytoskeletal attachment structures (adherens junctions, desmosomes, and focal adhesions). We review the existing literature on this topic and highlight outstanding questions and potential future directions that should clarify the impact of NDPK on the different cytoskeletal systems.
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http://dx.doi.org/10.1007/s00210-014-1046-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308521PMC
February 2015

Post-translational modifications of intermediate filament proteins: mechanisms and functions.

Nat Rev Mol Cell Biol 2014 Mar;15(3):163-77

1] Departments of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. [2] Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. [3] Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, Michigan 48109, USA.

Intermediate filaments (IFs) are cytoskeletal and nucleoskeletal structures that provide mechanical and stress-coping resilience to cells, contribute to subcellular and tissue-specific biological functions, and facilitate intracellular communication. IFs, including nuclear lamins and those in the cytoplasm (keratins, vimentin, desmin, neurofilaments and glial fibrillary acidic protein, among others), are functionally regulated by post-translational modifications (PTMs). Proteomic advances highlight the enormous complexity and regulatory potential of IF protein PTMs, which include phosphorylation, glycosylation, sumoylation, acetylation and prenylation, with novel modifications becoming increasingly appreciated. Future studies will need to characterize their on-off mechanisms, crosstalk and utility as biomarkers and targets for diseases involving the IF cytoskeleton.
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http://dx.doi.org/10.1038/nrm3753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079540PMC
March 2014

Reply: To PMID 23729294.

Hepatology 2014 Aug 18;60(2):767-8. Epub 2014 Jun 18.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI.

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http://dx.doi.org/10.1002/hep.26952DOI Listing
August 2014

A conserved rod domain phosphotyrosine that is targeted by the phosphatase PTP1B promotes keratin 8 protein insolubility and filament organization.

J Biol Chem 2013 Oct 3;288(43):31329-37. Epub 2013 Sep 3.

From the Departments of Molecular and Integrative Physiology and.

Post-translational modifications are important functional determinants for intermediate filament (IF) proteins. Phosphorylation of IF proteins regulates filament organization, solubility, and cell-protective functions. Most known IF protein phosphorylation sites are serines localized in the variable "head" and "tail" domain regions. By contrast, little is known about site-specific tyrosine phosphorylation or its implications on IF protein function. We used available proteomic data from large scale studies to narrow down potential phospho-tyrosine sites on the simple epithelial IF protein keratin 8 (K8). Validation of the predicted sites using a pan-phosphotyrosine and a site-specific antibody, which we generated, revealed that the highly conserved Tyr-267 in the K8 "rod" domain was basally phosphorylated. The charge at this site was critically important, as demonstrated by altered filament organization of site-directed mutants, Y267F and Y267D, the latter exhibiting significantly diminished solubility. Pharmacological inhibition of the protein-tyrosine phosphatase PTP1B increased K8 Tyr-267 phosphorylation, decreased solubility, and increased K8 filament bundling, whereas PTP1B overexpression had the opposite effects. Furthermore, there was significant co-localization between K8 and a "substrate-trapping" mutant of PTP1B (D181A). Because K8 Tyr-267 is conserved in many IFs (QYE motif), we tested the effect of the paralogous Tyr in glial fibrillary acidic protein (GFAP), which is mutated in Alexander disease (Y242D). Similar to K8, Y242D GFAP exhibited highly irregular filament organization and diminished solubility. Our results implicate the rod domain QYE motif tyrosine as an important determinant of IF assembly and solubility properties that can be dynamically modulated by phosphorylation.
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http://dx.doi.org/10.1074/jbc.M113.502724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829444PMC
October 2013

CD73 (ecto-5'-nucleotidase) hepatocyte levels differ across mouse strains and contribute to mallory-denk body formation.

Hepatology 2013 Nov 26;58(5):1790-800. Epub 2013 Aug 26.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI.

Unlabelled: Formation of hepatocyte Mallory-Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein, p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of MDB-susceptible C57BL and MDB-resistant C3H mouse strains can illuminate MDB-associated pathways. Using both targeted and unbiased metabolomic analyses, we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in messenger RNA (mRNA) level, protein expression, and enzymatic activity of the adenosine-generating enzyme, ecto-5'-nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and nonalcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB-inducing agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild-type (WT) and CD73(-/-) mice a DDC-containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73(-/-) , compared to WT livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was nearly absent in CD73(-/-) mice.

Conclusion: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. Expression of the adenosine-generating enzyme, CD73, contributes to experimental MDB induction and is highly regulated in MDB-associated liver injury in mice and in chronic human liver disease.
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http://dx.doi.org/10.1002/hep.26525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796030PMC
November 2013

Lamin aggregation is an early sensor of porphyria-induced liver injury.

J Cell Sci 2013 Jul 2;126(Pt 14):3105-12. Epub 2013 May 2.

Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.
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http://dx.doi.org/10.1242/jcs.123026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711202PMC
July 2013

Glucose and SIRT2 reciprocally mediate the regulation of keratin 8 by lysine acetylation.

J Cell Biol 2013 Feb 28;200(3):241-7. Epub 2013 Jan 28.

Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Lysine acetylation is an important posttranslational modification that regulates microtubules and microfilaments, but its effects on intermediate filament proteins (IFs) are unknown. We investigated the regulation of keratin 8 (K8), a type II simple epithelial IF, by lysine acetylation. K8 was basally acetylated and the highly conserved Lys-207 was a major acetylation site. K8 acetylation regulated filament organization and decreased keratin solubility. Acetylation of K8 was rapidly responsive to changes in glucose levels and was up-regulated in response to nicotinamide adenine dinucleotide (NAD) depletion and in diabetic mouse and human livers. The NAD-dependent deacetylase sirtuin 2 (SIRT2) associated with and deacetylated K8. Pharmacologic or genetic inhibition of SIRT2 decreased K8 solubility and affected filament organization. Inhibition of K8 Lys-207 acetylation resulted in site-specific phosphorylation changes of K8. Therefore, K8 acetylation at Lys-207, a highly conserved residue among type II keratins and other IFs, is up-regulated upon hyperglycemia and down-regulated by SIRT2. Keratin acetylation provides a new mechanism to regulate keratin filaments, possibly via modulating keratin phosphorylation.
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http://dx.doi.org/10.1083/jcb.201209028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563689PMC
February 2013

Oxidative stress, Nrf2 and keratin up-regulation associate with Mallory-Denk body formation in mouse erythropoietic protoporphyria.

Hepatology 2012 Jul 25;56(1):322-31. Epub 2012 Apr 25.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Unlabelled: 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 Fech(m1Pas) 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.
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http://dx.doi.org/10.1002/hep.25664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389581PMC
July 2012

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

J Cell Biol 2011 Oct;195(2):217-29

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA.

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.
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http://dx.doi.org/10.1083/jcb.201102142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198167PMC
October 2011

Anandamide oxidation by wild-type and polymorphically expressed CYP2B6 and CYP2D6.

Drug Metab Dispos 2011 May 2;39(5):782-8. Epub 2011 Feb 2.

Department of Pharmacology, University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA.

Anandamide is an arachidonic acid-derived endogenous cannabinoid that regulates normal physiological functions and pathophysiological responses within the central nervous system and in the periphery. Several cytochrome P450 (P450) isoforms metabolize anandamide to form hydroxylated and epoxygenated products. Human CYP2B6 and CYP2D6, which are expressed heterogeneously throughout the brain, exhibit clinically significant polymorphisms and are regulated by external factors, such as alcohol and smoking. Oxidative metabolism of anandamide by these two P450s may have important functional consequences for endocannabinoid system signaling. In this study, we investigated the metabolism of anandamide by wild-type CYP2B6 (2B6.1) and CYP2D6 (2D6.1) and by their common polymorphic mutants 2B6.4, 2B6.6, 2B6.9, and 2D6.34. Major differences in anandamide metabolism by the two isoforms and their mutants were found in vitro with respect to the formation of 20-hydroxyeicosatetraenoic acid ethanolamide (20-HETE-EA) and 14,15-epoxyeicosatetraenoic acid ethanolamide (14,15-EET-EA). Pharmacological studies showed that both 20-HETE-EA and 14,15-EET-EA bind to the rat brain cannabinoid CB1 receptor with lower affinities relative to that of anandamide. In addition, both products are degraded more rapidly than anandamide in rat brain homogenates. Their degradation occurs via different mechanisms involving either fatty acid amide hydrolase (FAAH), the major anandamide-degrading enzyme, or epoxide hydrolase (EH). Thus, the current findings provide potential new insights into the actions of inhibitors FAAH and EH, which are being developed as novel therapeutic agents, as well as a better understanding of the interactions between the cytochrome P450 monooxygenases and the endocannabinoid system.
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http://dx.doi.org/10.1124/dmd.110.036707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082373PMC
May 2011

Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat.

Gastroenterology 2011 Feb 9;140(2):627-637.e4. Epub 2010 Nov 9.

Department of Internal Medicine, University of Michigan, 1150 W Medical Center Drive, MSRB III, Room 9315, Ann Arbor, Michigan 48109, USA.

Background & Aims: Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia.

Methods: Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content.

Results: Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2.

Conclusions: These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.
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http://dx.doi.org/10.1053/j.gastro.2010.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031761PMC
February 2011

Keratin hypersumoylation alters filament dynamics and is a marker for human liver disease and keratin mutation.

J Biol Chem 2011 Jan 9;286(3):2273-84. Epub 2010 Nov 9.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-5622, USA.

Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.
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http://dx.doi.org/10.1074/jbc.M110.171314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023522PMC
January 2011

Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

PLoS One 2010 Oct 25;5(10):e13538. Epub 2010 Oct 25.

Department of Medicine, Stanford University School of Medicine, Palo Alto, California, United States of America.

Background: Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.

Methodology/principal Findings: In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.

Conclusions/significance: Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013538PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963603PMC
October 2010