Edward A. Doisy Department of
Biochemistry and Molecular Biology

Primary Faculty

Education
Ph.D., 2001, Washington University in St. Louis
Research Interests
The regulation of pre-mRNA processing, critical in the control of gene function and often linked to human disease, is mediated through the interaction between mRNA and protein partners. The TAR DNA binding protein (TDP-43) is an essential RNA binding protein whose dysfunction and aggregation are tightly associated with neurodegenerative disorders, i.e., amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, and frontotemporal dementia (FTLD). We investigate the cellular function and molecular structure of RNA binding proteins, such as TDP-43, to understand basic mechanisms of RNA processing and pathogenesis.
Recent Publications
  • TDP-43 regulates its mRNA levels through a negative feedback loop.
    Ayala YM, DeConti L, et al. (2011) EMBO J. 30:277-88.
  • Functional mapping of the interaction between TDP-43 and nRNP A2 in vivo.
    D-Ambrogio A, Buratti E, et al. Nucleic Acids Res. (2009) 37:4116-4126.
  • TDP-43 regulates pRB phosphorylation through the repression of CDK6 expression.
    Ayala YM, Misteli T and Baralle FE. Proc Natl Acad Sci USA. (2008) 105:3785-89.

Education
Ph.D., 2002, University of Barcelona
Research Interests
Our laboratory is interested in sterol homeostasis and in the molecular mechanisms involved in the conversion of macrophages into foam cells. This latter process is particularly relevant in several human pathologies, including atherosclerosis and different pulmonary lipidosis syndromes.
Recent Publications
  • miRNAs and high-density lipoprotein metabolism.
    Baldan A and de Aguiar Vallim TQ. Biochim Biophys Acta. (2016) pii: S1388.
  • miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice.
    Goedecke L, Rotllan N, et al. Atherosclerosis. (2015) 243(2):499-509.
  • Akt-mediated FoxO1 inhibition is required for liver regeneration.
    Pauta M, Rotllan N, et al. Hepatology. (2015) 63(5):1660-1674.
  • CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.
    Langhi C, Baldán A. Hepatology. (2015) 61(4):1227-1238.
Education
Ph.D., 1987, California Institute of Technology
Research Interests
Our lab is interested in understanding how two distinct eukaryotic methionine aminopeptidases (MetAPs) function in the amino-terminal processing of eukaryotic proteins and its role in angiogenesis. Recently, the type-2 MetAP was found to be the molecular target for angiogenesis inhibitors, TNP-470 and ovalicin. Angiogenesis is the process of new blood vessel formation. It plays very important roles in both physiological states and a variety of pathological states.
Recent Publications
  • Protein O-GlcNAcylation.
    Tian L, Simmons G, Chang YH. JSM Biochem Mol Biol. (2013) 1(1):1005-7.
  • A pilot study of pepsin in tracheal and oral secretions.
    Challom M, Tricomi S, et al. Am J Crit Care. (2013) 22(5):408-11.
Education
Ph.D., 2005, Texas A&M University
Research Interests
Immune cells have direct and indirect interaction with other cells. The indirect interactions include the secretion of small protein ligands such as cytokines, chemokines, and growth factors for binding and activating receptors. However, the molecular details of the binding mechanisms are still largely unknown. We are interested in understanding how these protein ligands interact with different receptors. We subsequently want to translate this information to develop small molecules modulating the protein ligands in a receptor-specific manner with therapeutic effects against inflammatory diseases and cancer. We use a variety of techniques, including biochemical and biophysical methods such as X-ray crystallography and NMR, high-throughput screening, virtual docking, biosensor-based label-free cellular assay system, as well as animal models of human diseases.
Recent Publications
  • New insights into mechanisms and functinos of chemokine (C-X-C motif) receptor 4 heteromerization in vascular smooth muscle.
    Evans AE, Tripathi A, et al. Int J Mol Sci. (2016) Jun 20 [Epub ahead of print].
  • Optogenetic control of chemokine receptor signal and T-cell migration.
    Xu Y, Hyun YM, et al. Proc Natl Acad Sci USA. (2014) 111(17):6371-6376.
Education
M.D., 1985, Catholic University School of Medicine, Rome, Italy.
Research Interests
We are interested in the structure, function and engineering of trypsin-like proteases and their zymogen forms. The main focus of the lab is on thrombin and prothrombin as key components of the blood coagulation system. Our experimental approach includes kinetics, thermodynamics, site-directed mutagenesis, X-ray structural biology and single molecule spectroscopy.
Recent Publications
  • Potassium and the K+/H+ exchanger Kha1p promote binding of copper to ApoFet3p multi-copper ferroxidase.
    Wu X, Kim H, et al. J Biol Chem. (2016) 291(18):9796-9806.
  • Data publication with the structural biology data grid supports live analysis.
    Meyer PA, Socias S, et al. Nat Commun. (2016) 7:10882.
  • How the linker connecting the two Kringles influences activation and conformational plasticity of prothrombin.
    Pozzi N, Chen Z, et al. J Biol Chem. (2016) 291(12):6071-6082.
  • Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold.
    Vogt AD, Chakraborty P, Di Cera E. J Biol Chem. (2015) 290(37):22435-22445.
  • John A. Schellman, 1924-2014.
    Di Cera E. Biophys Chem. (2015) pii:S0301.
  • Why ser and not thr brokers catalysis in the trypsin fold.
    Pelc LA, Chen Z, et al. Biochemistry. (2015) 54(7):1457-64.
Education
Ph.D., 1980, University of Tennessee Oakridge National Laboratory
Research Interests
We use Drosophila molecular genetics to understand how chromosome structure controls gene expression during development. Our studies have shed light on the molecular mechanisms of Cornelia de Lange syndrome, which causes diverse developmental deficits in humans.
Recent Publications
  • Drosophila nipped-B mutants model Cornelia de Lange syndrome in growth and behavior.
    Wu Y, Gause M, et al. PLoS Genet. (2015) 11(11):e1005655.
  • Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesion complex: Abstracts form he 2014 Scientific and Educational Symposium.
    Kline AD, Calof AL, et al. Am J Med Genet. (2015) 167(6):1179-1192.
  • Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.
    Izumi K, Nakato R, et al. Nat Genet. (2015) 47(4):338-344.
Significant Publications as an Independent Investigator

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila.


Patrick Morcillo, Christina Rosen, Mary K. Baylies, and Dale Dorsett.


Genes Dev. 11(20):2729-2740, 1997. (PMID 9334334)



This paper describes the cloning and functional characterization of the Chip/Ldb1 gene that is essential for the activity of many diverse transcriptional enhancers. Although our laboratory no longer actively works on this gene and protein, this paper stimulated several other laboratories to study its functions in diverse developmental and differentiation processes, including hematopoiesis, neurological development, and leukemia.

Model for regulation of cut by the remote wing margin enhancer and the sd, mam, and Chip genes.




Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes.


Robert A. Rollins, Patrick Morcillo and Dale Dorsett.


Genetics. 152(2):577-593, 1999. (PMID 10353901)



This paper described the genetic discovery, cloning and characterization of the Nipped-B gene that supports both sister chromatid cohesion and unexpectedly, the mechanisms that support long-range gene activation. This has turned out to be important in development, human genetic diseases, and cancer. It forms the basis of most of our laboratories current work, and the functions of this gene and protein are now intensively studied by dozens of laboratories around the world.

Dominant enhancement of the ct53d partial cut cause bithorax phenotypes in combination with heterowing phenotype by some Nipped mutations.




Association of cohesin and Nipped-B with transcriptionally active regions of the Drosophila melanogaster genome.


Ziva Misulovin, Yuri B. Schwartz, Xiao-Yong Li, Tatyana G. Kahn, Maria Gause, Stewart MacArthur, Justin C. Fay, Michael B. Eisen, Vincenzo Pirrotta, Mark D. Biggin, and Dale Dorsett.


Chromosoma. 117(1):89-102, 2008. (PMID 17965872)



This paper reports the first genome-wide analysis of the chromosome binding of the Nipped-B protein and cohesin complex essential for sister chromatid cohesion and chromosome segregation in a metazoan organism. The key finding is that these proteins only bind to genes when they are actively transcribed, and primarily to a subset of genes that are critical for growth and development. These findings form the basis of our current efforts to determine the mechanisms by which these proteins facilitate regulation of transcription and development. We reported multiple mechanisms that we have discovered, including support of enhancer-promoter communication and control of gene silencing, in subsequent papers.

Binding of Nipped-B, cohesin subunits, and PolII to a 2 Mb region of chromosome 3L. This region was chosen to illustrate typical features of cohesin and Nipped-B binding patterns seen throughout the genome.




Education
Ph.D., 1982, University of North Carolina, Chapel Hill
Research Interests
Research in my lab concerns four aspects of transcriptional regulation: histone biotinylation and gene expression; transcriptional activation and chromatin remodeling; RNA polymerase elongation factors and gene regulation; and heterochromatin and gene regulation. We use the fruit fly, Drosophila melanogaster, as a model to study mechanisms of gene activation and gene silencing.
Recent Publications
  • More than meets the eye: eye color and alcoholism.
    Eissenberg JC. Mo Med. (2016) 113:98-103.
  • Chapter 9: Medical Epigenetics and Twins.
    Eissenberg JC. in "Medical Epigenetics". (2016) Tollefsbol T, ed. (Elsevier), pp. 147-158.
  • Tying up loose ends: telomeres, genomic instability and lamins.
    Gonzalo S, Eissenberg JC. Curr Op Genet Dev. (2016) 37:109-118.
  • Different pathways to the lysosome: Sorting out alternatives.
    Hasanagic M., Waheed A, Eissenberg JC. Int Rev Cell Mol Biol. (2015) 320:75-101.
  • The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster.
    Hasanagic M., van Meel S., et al. Biol Open. (2015) 4:1316-1325.
Significant Publications as an Independent Investigator

Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster.


Joel C. Eissenberg, Tharappel C. James, Dawn M. Foster-Hartnett, Thomas Hartnett, Vivian Ngan, and Sarah C.R. Elgin.


Proc. Natl. Acad. Sci. USA. 87(24):9923-9927, 1990 (PMID 2124708).



This paper provided the first link between Heterochromatin Protein 1 (HP1) and gene silencing by heterochromatic position-effect gene silencing. The HP1 protein family is conserved from yeast to humans. This paper was a landmark in our understanding of how distinct forms of chromatin are organized in eukaryotes. It currently has 406 citations on Web of Science.




Genomic map of the HP-1 gene showing the relative annealing positions of synthetic oligonucleotides used in these studies.




Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila.


Brett Y. Lu, Peter C.R. Emtage, Brenda J. Duyf, Arthur J. Hilliker, and Joel C. Eissenberg.


Genetics. 155(2):699-708, 2000 (PMID 10835392).



This paper was the first to show that HP1, a protein previously associated with silencing of euchromatic genes by heterochromatin, is required for the normal expression of genes in heterochromatin. This research provided the genetic foundation for the current model that HP1 family proteins are chromatin organizing proteins, not silencing proteins. It currently has 114 citations on Web of Science.

Loss of heterochromatic silencing of a euchromatic gene in Su(var)2-5 mutant larvae.




dELL, an essential RNA polymerase II elongation factor with a general role in development.


Joel C. Eissenberg, Jiyan Ma, Mark A. Gerber, Alan Christensen, James A. Kennison, and Ali Shilatifard.


Proc. Natl. Acad. Sci. USA. 99(15):9894-9899, 2002. (12096188).



This paper was the first to describe the genetics of the RNA Polymerase II elongation factor, ELL. The ELL protein family is conserved from yeast to humans, and is implicated in leukemia and in HIV latency. It currently has 47 citations on Web of Science.

Models of ELL function in vivo.




Education
Ph.D., 1985, University of Missouri-Columbia
Research Interests
We are interested in biochemical mechanisms responsible for the pathophysiological sequelae of cardiovascular diseases including ischemic heart disease and atherosclerosis. Areas of research focus on enzymic and free radical targeting of membrane phospholipids, alterations in lipid metabolism, and alterations in signaling pathways as mechanisms involved in cardiovascular diseases. We combine our expertise using physiological models of disease coupled with expertise in mass spectrometry and bioorganic techniques to reveal new mechanistic insights into cardiovascular disease.
Recent Publications
  • Intestinal phospholipid remodeling is required for dietary lipid uptake and survival on a high-fat diet.
    Wang B, Rong X, et al. Cel Metab. (2016) Jan 27 [Epub ahead of print].
  • Akt-mediated FoxO1 inhibition is required for liver regeneration.
    Pauta M, Rotllan N, et al. Hepatology. (2015) Oct 16 [Epub ahead of print].
  • Identification of glutathione adducts of alpha-chlorofatty aldehydes produced in activated neutrophils.
    Duerr MA, Aurora R, Ford DA. J Lipid Res. (2015) 56(5):1014-1024.
  • Lpcat3-dependent production of arachidonoyl phospholipids is a key determinant of triglyceride secretion.
    Rong X, Wang B, et al. Elife. (2015) 4:e06557.
  • Dipeptidyl peptidase-4 inhibition ameliorates western diet-induced hepatic steatosis and insulin resistance through hepatic lipid remodeling and modulation of hepatic mitochondrial function.
    Aroor AR, Habibi J, et al. Diabetes. (2015) 64(6):1988-2001.
  • PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis.
    Shih DM, Yu JM, et al. FASEB J. (2015) 29(4):1185-1197.
Significant Publications as an Independent Investigator

Calcium-independent phospholipase A2 mediates CREB phosphorylation and c-fos expression during ischemia.


Scott D. Williams and David A. Ford.


Am. J. Physiol. 281(1): H168-176, 2001. (PMID 11406482)



This publication was the culmination of a number of studies in our lab focusing on the role of the calcium-independent phospholipase A2 (iPLA2) as a mediator of alterations in myocardium following myocardial ischemia and was the final publication of the first graduate student in my lab at SLU. This paper linked iPLA2 to nuclear signaling via PKA in the ischemic-reperfused heart. We shifted our focus from phospholipases to HOCl mediated signaling mechanisms after these stellar studies. This field is open to further investigation in my lab.

Proposed mechanisms mediating CREB phosphorylation in response to myocardial ischemia. LPC, choline-containing lysoglycerophospholipids (i.e., lysoplasmenylcholine and lysophosphatidylcholine).




Metabolism of myeloperoxidase-derived 2-chlorohexadecanal.


Kristin R. Wildsmith, Carolyn J. Albert, Dhanam S. Anbukumar, and David A. Ford.


J. Biol. Chem. 281(25):16849-16860, 2006 (PMID 16611638)



Our laboratory discovered that plasmalogens are targeted by hypochlorous acid, which is produced by the activity of myeloperoxidase in activated neutrophils, monocytes and macrophages. While the first paper describing this pathway was significant, this publication resulting from the tremendous work led by graduate student, Kristin Wildsmith, and Lab Manager, Carolyn Albert, with the aide of graduate student, Dhanam Subramanian Anbukumar, showed the many chlorinated lipid species that could be produced as a result of the reaction of plasmalogens and hypochlorous acid. The work from these studies introduced us to the chlorinated lipidome. The study of these lipids and their role in ischemia/reperfusion, atherosclerosis, sepsis and infectious disease remains an evolving focus of our lab.




Alpha-Chlorofatty acid accumulates in activated monocytes and causes apoptosis through reactive oxygen species production and endoplasmic reticulum stress.


Wen-yi Wang, Carolyn J. Albert, and David A. Ford.


Arterioscler. Thromb. Vasc. Biol. 34(3):523-532, 2014. (PMID 24371082)



Post-doc Wen-yi Wang showed the amounts of alpha-chlorofatty acid produced in human monocytes and then revealed the mechanism by which these lipids elicit monocyte apoptosis through the production of hydrogen peroxide and ER stress. Furthermore, showing these pathways are active in immortalized cell lines, such as the human monocytic cell line, THP-1, has provided our lab with a useful tool to further examine mechanisms and receptors through which chlorinated lipids elicit their cellular effects. This paper illustrates a current area of investigation in the lab.

2-ClHA induces apoptosis in primary human monocytes, THP-1 monocytes, and RAW 264.7 macrophages.




Education
Ph.D., Washington University School of Medicine
Research Interests
The human genome is organized into different levels of complexity. Packaging of DNA into different chromatin states and 3D nuclear organization of the genome are emerging as additional levels of regulation of genome function. Our broad research interests are to understand how alterations of nuclear architecture, chromatin structure, and genome stability contribute to the processes of aging and cancer. Our studies revealed that the structural nuclear proteins A-type lamins play a key role in the maintenance of telomere structure, length and function, as well as mechanisms of DNA double-strand break repair. Specifically, loss of A-type lamins increases the levels of the protease cathepsin L and its entry into the nucleus, which in turn leads to degradation of proteins with important roles in cell cycle regulation -Rb family members- and DNA repair -53BP1-. Loss of A-type lamins also leads to repression of BRCA1 and RAD51 genes, critical factors in homologous recombination. Interestingly, inhibition of cathepsin L activity with vitamin D or specific inhibitors rescues some of the phenotypes of lamins-deficient cells, providing new therapeutic possibilities. Most recently, we found that these novel pathways are also activated in BRCA1-deficient cells and subsets of breast cancer patients. Our current work aims to characterize in detail how these pathways contribute to the pathophysiology of cancer, aging, and laminopathies with the ultimate goal of using them as potential biomarkers for diagnosis, prognosis, and customization of treatment.
Recent Publications
  • Methods to minitor DNA repair defects and genomic instability in the context of a disrupted nuclear lamina.
    Gonzalo S, Kreienkamp R. Methods Mol Biol. (2016) 1411:419-437.
  • Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes.
    Kreienkamp R, Croke M, et al. Oncotarget. (2016) Apr 27 [Epub ahead of print].
  • Vitamin D/vitamin D receptor axis regulates DNA repair during oncogene-induced senescence.
    Graziano S, Johnston R, et al. Oncogene. (2016) Apr 4 [Epub ahead of print].
  • Tying up loose ends: telomeres, genomic instability and lamins.
    Gonzalo S, Eissenberg JC. Curr Op Genet Dev. (2016) 37:109-118.
  • Loss of lamin A function increases chromatin dynamics in the nuclear interior.
    Bronshtein I, Kepten E, et al. Nat Commun. (2015) 6:8044-8052.
  • DNA repair defects and genome instability in Hutchinson-Gilford Progeria Syndrome.
    Gonzalo S, Kreienkamp R. Curr Opin Cell Biol. (2015) 34:75-83.
Significant Publications as an Independent Investigator

BRCA1 loss activates cathepsin L-mediated degradation of 53BP1 in breast cancer cells.


David A. Grotsky, Ignacio Gonzalez-Suarez, Anna Novell, Martin A. Neumann, Sree C. Yaddanapudi, Monica Croke, Montserrat Martinez-Alonso, Abena B. Redwood, Sylvia Ortega-Martinez, Zhihui Feng, Enrique Lerma, Teresa Ramon y Cajal, Junran Zhang, Xavier Matias-Guiu, Adriana Dusso, and Susana Gonzalo.


J. Cell Biol. 200(2):187-202, 2013 (PMID 23337117)

Breast cancers classified as triple-negative (TNBC) and BRCA1-deficient are particularly aggressive and difficult to treat. A major breakthrough was the finding that these tumors are exquisitely sensitive to inhibitors of poly(ADP-ribose) polymerase (PARPi). Phase II clinical trials have shown encouraging outcomes with tolerable side effects. However, a significant fraction of these cancers acquire resistance. Elegant studies demonstrated that loss of the DNA repair protein 53BP1 contributes to the resistance of BRCA1-deficient cells and tumors to PARPi. Thus, raising the levels of 53BP1 in these aggressive tumors could potentially restore their sensitivity to PARPi and other genotoxic agents.

In this study, we uncovered a molecular mechanism regulating 53BP1 levels that can be targeted for therapeutic purposes. We demonstrated that BRCA1 loss activates cathepsin L (CTSL)–mediated degradation of 53BP1, rescuing homologous recombination repair and allowing BRCA1-deficient cells to overcome genomic instability and growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and PARPi, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor (VDR), and 53BP1 emerged from this study as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identified here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.

Model of functional relationship between vitamin D/VDR axis and DNA repair factors during Ras-induced senescence.




Vitamin D/vitamin D receptor axis regulates DNA repair during oncogene-induced senescence.


Simona Graziano, Rachel Johnston, O. Deng, Junran Zhang, and Susana Gonzalo.


Oncogene. Apr 4 [Epub ahead of print], 2016. (PMID 27041576)

Oncogenic Ras expression is associated with activation of the DNA damage response (DDR) pathway, as evidenced by elevated DNA damage, primarily DNA double-strand breaks (DSBs), and activation of DNA damage checkpoints, which in primary human cells leads to entry into senescence. DDR activation is viewed as a physiological barrier against uncontrolled proliferation in oncogenic Ras-expressing cells, and arises in response to genotoxic stress due to the production of reactive oxygen species (ROS) that damage DNA, and to hyper-replication stress. Although oncogene-induced senescence (OIS) is considered a tumor suppressor mechanism, the accumulation of DNA damage in senescent cells is thought to cause genomic instability, eventually allowing secondary hits in the genome that promote tumorigenesis. To date, the molecular mechanisms behind DNA repair defects during OIS remain poorly understood.

Our study shows that oncogenic Ras expression in human primary cells results in down-regulation of BRCA1 and 53BP1, two key factors in DNA DSBs repair by homologous recombination (HR) and non-homologous end joining (NHEJ), respectively. As a consequence, Ras-induced senescent cells are hindered in their ability to recruit BRCA1 and 53BP1 to DNA damage sites. While BRCA1 is down-regulated at transcripts levels, 53BP1 loss is caused by activation of cathepsin L (CTSL)-mediated degradation of 53BP1 protein. Moreover, we discovered a marked down-regulation of vitamin D receptor (VDR) during OIS, and a role for the vitamin D/VDR axis regulating the levels of these DNA repair factors during OIS. This study reveals a new functional relationship between the oncogene Ras, the vitamin D/VDR axis, and the expression of DNA repair factors, in the context of OIS. The observed deficiencies in DNA repair factors in senescent cells could contribute to the genomic instability that allows senescence bypass and tumorigenesis.

Model of functional relationship between vitamin D/VDR axis and DNA repair factors during Ras-induced senescence.




Vitamin D receptor signaling improves Hutchinson-Gilford Progeria Syndrome cellular phenotypes.


Ray Kreienkamp, Monica Croke, Martin A. Neumann, Gonzalo Bedia-Diaz, Simona Graziano, Adriana Dusso, Dale Dorsett, Carsten Carlberg, and Susana Gonzalo.


Oncotarget. In press, 2016.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence.

This study uncovers new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via vitamin D (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the vitamin D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. Vitamin D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging.




Education
Ph.D., 1986, Technical University of Wroclaw, Poland
Research Interests
Our lab has two major research interests: mechanisms of transcription regulation and development of novel sensors for biomolecule detection and imaging. Our interest in transcription regulation is to understand the mechanism of transcription initiation by bacterial and archaeal RNA polymerases. Our primary focus in sensor research is to develop robust highly specific and sensitive molecular sensors that could be utilized in research, medical diagnosis and pathogen detection.
Recent Publications
  • Real-time observation of backtracking by bacterial RNA polymerase.
    Lass-Napiorkowska A and Heyduk T. Biochemistry. (2016) 55:647-658. PubMed PMID: 26745324
  • Next generation sequencing-based analysis of RNA polymerase functions.
    Heyduk T and Heyduk E. Methods. (2015) 86:37-44. PubMed PMID: 25937393
  • Ribosome display enhanced by next generation sequencing: A tool to identify antibody-specific peptide ligands.
    Heyduk T and Heyduk E. Anal Biochem. (2014) 464:73-82. PubMed PMID: 25058925
  • Kinetics of promoter escape by bacterial RNA polymerase: effects of promoter contacts and transcription bubble collapse.
    Ko J, Heyduk T. Biochem J. (2014) 463(1):135-144. PubMed PMID: 24995916
  • Next generation sequencing-based parallel analysis of melting kinetics of 4096 variants of a bacterial promoter.
    Heyduk E, Heyduk T. Biochemistry. (2014) 53(2):282-292. PubMed PMID: 24359527
Education
Ph.D., 1972, National Taiwan University
Research Interests
The areas of research in this laboratory are: 1) autocrine transformation by the v-sis/c-sis oncogene and novel trans-Golgi network (TGN) signal transduction, and 2) role of the transforming growth factor ß (TGF-ß) type V receptor (TR-V) in the biological functions of TGF-ß.
Recent Publications
  • CRSBP-1/LYVE-1 ligands stimulate contraction of the CRSBP-1 associated ER network in lymphatic endothelial cells.
    Hou WH, Liua IH, et al. FEBS Lett. (2012) 586(10):1480-7.
Education
Ph.D., 1972, University of California, San Francisco
Research Interests
The primary goal of this research is to develop a new chemotherapeutic option for the treatment of cancer. We have chosen to develop Zn as a chemotherapeutic agent, based on our observations that Zn treatment of a variety of cancer cell lines results cell death. The rapidity with which cell death is induced is very attractive with respect to treatment protocols and is in dramatic contrast to the days-weeks required to achieve cell death with current chemotherapeutics. Importantly, Zn-mediated cell death is independent of the presence or absence of growth factors, is not limited to rapidly growing cells, and is independent of p53 status.
Recent Publications
  • Zinc is a potential therapeutic for chemoresistant ovarian cancer.
    Bastow M, Kriedt CL, et al. J Exp Ther Oncol. (2011) 9(3):175-181.
  • Zinc functions as a cytotoxic agent for prostate cancer cells independent of culture and growth conditions.
    Kriedt CL, Baldassare J, et al. J Exp Ther Oncol. (2010) 8:287-295.
Education
Ph.D., 1993 Engelhardt Institute of Biochemistry and Molecular Biology, Moscow, Russian Academy of Sciences
Research Interests
Our lab studies the mechanism of protein function at the atomic resolution level utilizing X-ray crystallography and biochemical approaches. The main focus is recombination mediator proteins (RMPs), which are essential for genome stability and DNA repair in all organisms.
Recent Publications
  • Correction: High resolution crystal structure of human beta-glucuronidase reveals structural basis of lysosome targeting.
    Hassan MI, Waheed A, et al. PLoS One. (2015) 10(9):e0138401.
  • RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.
    Ryzhikov M, Gupta R, et al. J Biol Chem. (2014) 289(42):28846-28855.
  • Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors.
    Pandey KK, Bera S, et al. J Biol Chem. (2014) 289(28):19648-58.
Education
Ph.D., 2010, University of Padua, Italy.
Research Interests
Our laboratory studies the structure, function and dynamics of proteins that are mistakenly targeted by autoantibodies in autoimmune disorders such as Antiphosholipid Syndrome (APS), Systemic Lupus Erythematous (SLE) and Rheumatoid Arthritis (RA). Our aim is to understand the origin of the autoantibodies, how the antigen-antibody recognition occurs at the molecular level and what are the molecular pathways involved in the transduction of the signal. Our goal is to develop strategies to diagnose, manage or treat the onset and progression of the disease. To achieve our goals, we apply a unique combination of cell and molecular biology, protein engineering, X-ray crystallography, single molecule fluorescence spectroscopy (smFRET and FCS) and surface plasmon resonance (SPR).
Recent Publications
  • How the linker connecting the two Kringles influences activation and conformational plasticity of prothrombin.
    Pozzi N, Chen Z, et al. J Biol Chem. (2016) Jan 12 [Epub ahead of print].
  • Why ser and not thr brokers catalysis in the trypsin fold.
    Pelc LA, Chen Z, et al. Biochemistry. (2015) 54(7):1457-64.
  • WEDGE: An anticoagulant thrombin mutant produced by autoactivation.
    Wood DC, Pelc LA, et al. J Thromb Haemost. (2014) 13(1):111-4.
  • Prothrombin structure: Unanticipated features and opportunities.
    Pozzi N, Di Cera E. Expert Rev Proteomics. (2014) 11(6):653-5.
  • The linker connecting the two kringles plays a key role in prothrombin activation.
    Pozzi N, Chen Z, et al. Proc Natl Acad Sci USA. (2014) 111(21):7630-5.
  • Essential role of conformational selection in ligand binding.
    Vogt AD, Pozzi N, et al. Biophys Chem. (2014) 186:13-21.
Education
Ph.D., 1989, Boston University
Research Interests
Our research is primarily focused on understanding the mechanism by which coagulation proteases interact with their target cofactors, substrates, and inhibitors, and how heparin enhances the inhibitory function of antithrombin in the regulation of the proteolytic activities of these proteases. Another project in the lab focuses on understanding the mechanism by which coagulation proteases interact with endothelial cell surface receptors to elicit diverse intracellular signaling responses. We employ biophysical, biochemical, and molecular biological approaches to study these questions.
Recent Publications
  • Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.
    Liang HP, Kerschen EJ, et al. Blood. (2015) 126(21):2415-2423.
  • Expression and characterization of Gly-317 variants of Factor IX causing variable bleeding in Hemophilia B patients.
    Lu Q, Yang L, et al. Biochemistry. (2015) 54(24):3814-3821.
  • Inorganic polyphosphate elicits pro-inflammatory responses through activation of the mammalian target of rapamycin complexes 1 and 2 in vascular endothelial cells.
    Hassanian SM, Dinarvand P, et al. J Thromb Haemost. (2015) 13(5):860-871.
  • Protein C Thr315Ala variant results in gain-of-function but manifests as type II deficiency in diagnostic assays.
    Ding Q, Yang L, et al. Blood. (2015) 125(15):2428-2434.
  • Intraperitoneal administration of activated protein C prevents post surgical adhesion band formation.
    Dinarvand P, Hassanian SM, et al. Blood. (2015) 125(8):1339-1348.
Education
M.D., 1981, Ph.D., 1984, University of Illinois at Urbana-Champaign
Research Interests
Our clinical activities include screening for inborn errors of metabolism in children by quantifying chemicals in body fluids by gas chromatography-mass spectrometry. We developed a new method of sample preparation which allows carbohydrates and amino and organic acids to be detected in the same sample. Our current research is concerned with the evaluation of special nutritional needs in children with Down syndrome and the diagnosis of vitamin deficiency by quantitation of urinary metabolites after an oral dose of amino acids and other food constituents.
Recent Publications
  • Dicarboxylic acid excretion in normal formula-fed and breastfed infants.
    Anderson M, Eliot K, et al. Nutr Clin Pract. (2016) May 6 [Epub ahead of print].
  • Estimation of glucose utilization in a type 2 diabetes mellitus patient on insulin analogs with tumor hypoglycemia induced by IGF-II.
    Code S, Albert SG, et al. Growth Horm IGF Res. (2016) 26:8-10.
  • Urinary organic acids quantitated in a healthy north Indian pediatric population.
    Kumari C, Singh A, et al. Indian J Clin Biochem. (2015) 30(2):221-9.
Significant Publications as an Independent Investigator

Misidentification of propionic acid as ethylene glycol in a patient with methylmalonic acidemia.


James D. Shoemaker, Robert E. Lynch, Joseph W. Hoffmann, and William S. Sly.


J. Pediatr. 120(3):417-421, 1992 (PMID 1538288).

My first significant publication was the result of my involvement in the case of a mother, who was initially incarcerated on charges of murder because high levels of ethylene glycol were present in her child's blood. However, upon further analysis by the Metabolic Screening Laboratory, the child was found to have methylmalonic acidemia, an inborn metabolic disorder resulting in increased propionic acid, which can be mistaken for ethylene glycol by the chromatographic techniques commonly used by clinical laboratories. Our analysis, which used combined gas chromatography-mass spectrometry techniques, was able to distinguish between the products of ethylene glycol metabolism and those associated with metabolic abnormalities. As a result, the mother was exonerated of all charges.

The story can also be seen on YouTube (viewed over 3175 times): Deadly Formula.


Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria.


Coy D. Fitch, Guang-Zuan Cai, Yi-Feng Chen, James D. Shoemaker.


Biochim. Biophys. Acta 1454(1):31-37, 1999 (PMID 10354512).



My next significant publication is my most-cited, with 114 citations. Here I identified linoleic acid as the substance that promoted polymerization of ferriprotoporphyrin IX or FP. The ability of malaria parasites to sequester FP from hemozoin in an insoluble and non-toxic form is an important adaptation, which allows them to digest hemoglobin while avoid FP toxicity. The research was done in collaboration with Coy Fitch, M.D., of the Department of Internal Medicine at SLU.




One-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure.


James D. Shoemaker.


J. Vis. Exp. Jun 25(40):e2014, 2010 (20613709).




Urinary organic acids quantitated in a healthy north Indian pediatric population.


Chandrawati Kumari, Ankur Singh, Siddharth Ramji, James D. Shoemaker, Seema Kapoor.


Indian J. Clin. Biochem. 30(2):221-229, 2015 (25883433).

I choose two related publications for my final entry. The first is a video publication that explains how to do One Step Metabolomics, a procedure I introduced that has now been done over 20,000 times for diagnostic purposes in the Metabolic Screening Lab. It is significant because it provides a more specific test for screening of rare genetic disorders and identification of suspected inborn errors of metabolism. The "urease" process allows for removal of urea from the fluid being tested, permitting most other water soluble metabolites to be dehydrated and processed via chromatography and mass spectrometry. The method has been cited 91 times. The video can be viewed on the Jove site.

The second is my "favorite" publication because it shows the global impact of One Step Metabolomics as used by investigators in New Delhi, who invited me to speak there, and in Hyderabad, Bangalore, and Mumbai in 2012 and 2013, to promote screening for inborn errors using our method. It is significant because knowing the concentrations of organic acids in the general healthy pediatric population can aid in the correct diagnosis of metabolic abnormalities, since these values vary depending on environment, food consumption, and genotype, as well as other various factors.


Education
Ph.D., 1991, University of Gdansk, Poland
Research Interests
We are broadly interested in the role, function and regulation of the ubiquitin-proteasome system. Our former projects focused on the mechanism by which proteins are recruited for degradation by the 26S proteasome in yeast. We have recently initiated three new research directions that focus on the role of proteasomal proteolysis in 1) autoimmune diseases (type 1 diabetes); 2) protein misfolding diseases (liver disease associated with alpha 1 antitrypsin deficiency); and early antiviral responses (ectromelia virus/primary mouse macrophages). Biochemical and cellular approaches are our primary research tools.
Recent Publications
  • PiZ mouse liver accumulates polyubiquitin conjugates that associate with catalytically active 26S proteasomes.
    Haddock CJ, Blomenkamp K, Gautam M, James J, Mielcarska J, Gogol E, Teckman J, Skowyra D. PLoS One. (2014) 9(9):e106371.
  • Broad utility of an affinity-enrichment strategy for unanchored polyubiquitin chains.
    Strachan J, Shaw B, Tooth D, Krishna VG, de Pozo JC, Hill K, Bennett M, Gautam M, Skowyra D, Jacobson AD, Liu CW, Oldham N, Layfield R. J Proteomics Bioinform. (2013) S7:1-7.
  • Immunoproteasome activation during early antiviral response in mouse pancreatic β-cells by IFNβ: New insights into auto-antigen generation in Type I diabetes?
    Freudenburg W, Gautam M, Chakraborty P, James J, Richards J, et al. J Clin Cell Immunol. (2013) 4(2).
  • Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNbeta in mouse pancreatic beta-cells.
    Freudenburg W, Gautam M, Chakraborty P, Richards J, Salvatori A, Baldwin A, Schriewer J, Buller M, Corbett J, Skowyra D. PLOS One. (2013) 8(2):e52408.

Full list of publications in PubMed: Skowyra D
Significant Publications as an Independent Investigator

Release of ubiquitin-charged Cdc34-S~Ub from the RING domain is essential for ubiquitination of the SCFCdc4-bound substrate Sic1.


Andrew E Deffenbaugh, K. Matthew Scaglione, Lingxiao Zhang, Johnnie M. Moore, Tione Buranda, Larry A. Sklar, and Dorota Skowyra.


Cell. 114(5):611-622, 2003. (PMID 13678584)

The mechanism of polyubiquitination was initially thought to involve stable interaction between the E2, the E3 and a substrate. Our "hit and run" hypothesis (Figure below) published in Deffenbaugh et al. was the first to propose that, in contrast to stability of the E3-substrate interaction, the process of polyubiquitination involves repeated cycles of interaction between the E2 and the E3. In the same study, we also provided evidence suggesting that a release of ubiquitin-charged Cdc34 E2 from its primary recruitment site on the RING domain of SCFCdc4 could play a role in the process of polyubiquitination.




The "Hit and Run" model for Cdc34 function with SCFCdc4.



ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle.


Shalon E. Babbitt, Alexi Kiss, Andrew E. Deffenbaugh, Yie-Hwa Chang, Eric Bailly, Hediye Erdjument-Bromage, Paul Tempst, Tione Buranda, Larry A. Sklar, Jennifer Baumler, Edward Gogol, and Dorota Skowyra.


Cell. 121(4):553-565, 2005 (PMID 15907469)

Using the yeast 26S proteasomes co-purified with proteasome-interacting proteins (PIPs), such as polyubiquitinated substrates and the SCF E3 ubiquitin ligase complexes, we showed that the end of substrate proteolysis is linked to an ATP hydrolysis-dependent dissociation and disassembly of the 19S activator, and that this step co-incides with a burst-like release of product peptides. Based on these findings, we proposed the "chew and spew" model for coupling ATP hydrolysis with diassembly of the 26S proteasome (Figure below). This study was the first demonstration that disassembly of the 26S proteasome could be linked to the catalytic cycle.



The "Chew and Spew" model for coupling ATP hydrolysis to disassembly of the 26S proteasome.



Destabilization of binding to cofactors and SCFMet30 is a rate-limiting regulatory step in degradation of polyubiquitinated Met4 by the 26S proteasome.


Srikripa Chandrasekaran, Andrew E. Deffenbaugh, David A. Ford, Eric Bailly, Neal Mathias, and Dorota Skowyra.


Mol. Cell. 24(5):689-699, 2006. (PMID 17157252)

In Chandrasekaran et al., we reported a novel regulatory mechanism in which a substrate polyubiquitinated with the cannonical K48-type of polyubiquitin chain can collaborate with other proteins to temporarily stall its own degradation until receiving a further regulatory signal, This paradigm was established by reconstruction of the SCFMet30-mediated polyubiquitination and proteolysis of Met4, the transcriptional master regulator of genes involved in methionine biosynthesis in yeast (Figure below).



Roadmap highlighting the relationship between the SCFMet30-Met4 interplay and the regulatory schemes involved in sulfur assimilation, oxidative stress and cell division (Chandrasekaran and Skowyra, 2008.



Education
Ph.D., 1995, University of Padua, Italy
Research Interests
Our laboratory focuses on the mechanisms of DNA replication and repair, and on the possible strategies to target these mechanisms for cancer treatment. Aberrant DNA replication is one of the leading causes of mutations and chromosome rearrangements associated with several cancer related pathologies. At the same time, agents that stall or damage DNA replication forks are widely used for chemotherapy, in the attempt to selectively target highly proliferating cancer cells. Our work provides a new rationale to design novel molecularly-guided treatments targeting the pathways of replication stress response to cancer chemotherapeutics.
Recent Publications
  • Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.
    Yamamoto K, Wang J, et al. Elife. (2016) Jun 15 [Epub ahead of print].
  • Replication stress: getting back on track.
    Berti M and Vindigni A. Nat Struct Mol Biol. (2016) 23:103-109.
  • Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.
    Kenig S, Bedolla DE, et al. Biophys Chem. (2015) 207:90-96.
  • Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures.
    Pike ACW, Gomathinayagam S, et al. Proc Natl Acad Sci USA. (2015) 112(14):4286-4291.
  • Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.
    Zellweger R, Dalcher D, et al. J Cell Biol. (2015) 208(5):563-579.
  • DNA2 drives processing and restart of reversed replication forks in human cells.
    Thangavel S, Berti M, et al. J Cell Biol. (2015) 208(5):545-562.

Full list of publications in PubMed: Vindigni A
Significant Publications as an Independent Investigator

Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition.


Matteo Berti, Arnab Ray Chaudhuri, Saravanabhavan Thangavel, Shivasankari Gomathinayagam, Sasa Kenig, Marko Vujanovic, Federico Odreman, Timo Glatter, Simona Graziano, Ramiro Mendoza-Maldonado, Francesca Marino, Bojana Lucic, Valentina Biasin, Matthias Gstaiger, Ruedi Aebersold, Julia M Sidorova, Raymond J. Monnat Jr., Massimo Lopes, and Alessandro Vindigni.


Nat. Struct. Mol. Biol. 20(3):347-354, 2013. (PMID 23396353).



Replication fork reversal is rapidly emerging as a pivotal mechanism of replication stress response to cancer chemotherapeutics. This work identifies the first molecular mechanism required to restart replication forks that have reversed upon treatment with DNA topoisomerase I inhibitors. It also provides a new rationale to improve current chemotherapeutic modalities based on the use of DNA replication inhibitors. This article was rated as a "must read" by the Faculty of 1000.


PARP PARylation activity is not required to form reversed forks, but it promotes the accumulation of regressed forks by inhibiting RECQ1 fork restoration activity, thus preventing premature restart of regressed forks.




DNA2 drives processing and restart of reversed replication forks in human cells.


Saravanabhavan Thangavel, Matteo Berti, Maryna Levikova, Cosimo Pinto, Shivasankari Gomathinayagam, Marko Vujanovic, Ralph Zellweger, Hayley Moore, Eu Han Lee, Eric A. Hendrickson, Petr Cejka, Sheila Stewart, Massimo Lopes, and Alessandro Vindigni.


J. Cell Biol. 208(5):545-562, 2015 (PMID 25733713).



This work defines new important roles for different human nucleases in replication stress response to cancer chemotherapeutics and opens new avenues to study the link between nucleolytic processing of stalled replication intermediates and chemotherapeutic sensitivity.

Electron micrograph of a partially single-stranded (left) and entirely double-stranded (right) reversed fork observed on genomic DNA upon HU-treatment. The black arrow points to the ssDNA region on the reversed arm.
D, daughter strand; P, parental strand; R, reversed arm.




Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures.


Ashley C.W. Pike, Shivasankari Gomathinayagam, Paolo Swuec, Matteo Berti, Ying Zhang, Christina Schnecke, Francesca Marino, Frank von Delft, Ludovic Renault, Alessandro Costa, Opher Gileadi, and Alessandro Vindigni.


Proc. Natl. Acad. Sci. USA. 112(14):4286-4291, 2015. (25831490).



This work determines the first DNA complex structures of the human RECQ1 helicase. These structures provide new insight into the RecQ helicase mechanism of DNA tracking, strand separation, and Holliday junction branch migration. This work helps clarify how different RecQ enzymes are uniquely adapted to process potentially recombinogenic DNA structures that arise upon replication stress.

Overall structure of the RECQ1/DNA complex and trajectory of the ssDNA tail. Perpendicular view of isolated monomer/DNA showing the trajectory of DNA. The top and bottom strands of the tailed duplex are colored black and orange, respectively. A third ssDNA strand from an adjacent complex in the crystal, which base-pairs at the separation junction, is colored cyan.




Education
Ph.D., 2006, University of Wisconsin-Madison
Research Interests
Few biochemical reactions are as critical for life as translation. We are interested in understanding the functional consequences of arrested translation (or "ribosome stalling") for controlling gene expression and protein biogenesis using genome-wide proteomics, next-generation sequencing, bacterial genetics and biochemistry. We also aim to investigate the selectivity and resistance properties of antibiotics that target the ribosome tunnel. This work will help in the development of more effective antimicrobial drugs.
Recent Publications
  • Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation.
    Basu A, and Yap, MNF. Nucleic Acids Res. (2016) [Epub ahead of print].
  • Sequence selectivity of macrolide-induced translational attenuation.
    Davis AR, Gohara DW, Yap MN. Proc Natl Acad Sci USA. (2014) 111(43):15379-15384.