University of Nebraska–Lincoln

RESEARCH INTERESTS:

The main research topic the Alfano research group is interested in is the type III secretion system of Pseudomonas syringae and how it mediates the interaction this gram-negative bacterium has with plants.

RESEARCH DESCRIPTION:

My research interests are concerned with understanding how bacterial pathogens cause disease in plants and how their strategies differ from the strategies employed by bacterial pathogens of animals. Research in my laboratory primarily is focused on understanding a specialized protein secretion apparatus, called the type III secretion system, present in gram-negative bacterial pathogens of plants and animals. Type III systems secrete multiple virulence proteins, some of which are transferred directly into eukaryotic cells in a contact dependent manner. Acquisition of a type III secretion system appears to be a key adaptation that allowed many gram-negative bacteria to become pathogens – mutants with a disabled type III system are essentially nonpathogenic. My research group studies the type III secretion system present in the bacterial plant pathogen, Pseudomonas syringae. P. syringae is a leaf spotting pathogen whose various strains display host specificity: Different strains are only capable of causing disease in certain plants. We study the interactions of P. syringae with such crop plants as tobacco, soybeans, and tomato, as well as the interactions of P. syringae with the genetically amenable plant Arabidopsis. Studying the interaction of P. syringae and Arabidopsis. is particularly attractive because it allows us to relatively easily identify key molecular attributes of both the pathogen and the host with the long-term goal of understanding the intimacies involved in bacterial parasitism.

REPRESENTATIVE PUBLICATIONS:

RESEARCH DESCRIPTION:

Research on Anti-tuberculosis Drug Development and Biodefense: This research is focused on tuberculosis and the development of new drug therapies.  Re-mergence of tuberculosis is caused by multiple drug-resistant strains and the need to develop new therapies and other control strategies. In this aspect, in collaboration with Dr. Ofelia Chacon (University of Nebraska, and Corporacion de Investigaciones Biologicas, Medellin-Colombia), we have focused on D-alanine racemase and D-alanine ligase, the presumed lethal targets of D-cycloserine, an analog of D-alanine. This enzyme forms the critical dipeptide D-alanyl-D-alanine, an essential building block of peptidoglycan. This line of research was developed more recently, first via institutional grants to develop “proof of concept” in the model system Mycobacterium smegmatis. A recent grant from the National Institutes of Health has allowed us to further pursue the development of D-alanine ligase as a target for rational drug design. These studies resulted in various publications and the granting of a US patent.  More recently, we have developed collaborations with Dr. James Sacchettini (Department of Biochemistry and Biophysics, Texas A&M University) to study the crystal structure of the drug target.  In this context, on going research is focused on:

1. Determine the essential roles of D-alanine ligase and D-alanine racemase in the physiology of M. tuberculosis. 
2. Search for novel inhibitors of the M. tuberculosis D-alanine ligase that could serve as novel anti-tuberculosis drugs.
3. Expand drug target search to additional essential steps in peptidoglycan biosynthesis and D-alanine metabolism. such as L-alanine dehydrogenase.
4. Determine the mechanisms of redox homeostasis underlying tuberculosis
   latency.

Other:  Other research interests include Johne’s Disease and Mycobacterium paratuberculosis.  This slow-growing organism, which doubles the generation time of M. tuberculosis, is the agent of Johne’s disease. This chronic enteritis affects all ruminants and is a major cause of economic loss to the dairy industry that has been estimated to exceed 1.0 billion dollars per year.  Our studies on paratuberculosis were directed to establish the molecular genetics of this microorganism. My laboratory at the University of Nebraska was the first to develop a gene transfer system for M. paratuberculosis. Since then, we have expanded our approach to demonstrate the expression of reporter genes and their usefulness to track the microorganism and test drug susceptibility. I contributed to the DNA sequencing project, carried out at the University of Minnesota, by identifying the prototype strain to be sequenced, a proposal that was based on our prior observations regarding the feasibility of genetic manipulations for this strain. We have recently generated a complete mutant bank of this strain and identified mutants that are attenuated in bovine macrophages. These mutants are now being tested in animal models including baby goats (with Dr. Nahum Shpigel, Israel) and mice (with Dr. Luiz Bermudez, Oregon State), and we are currently in the preliminary steps to test the mutants in cattle.  Funding for this program is provided by various grants from the US Deaprtment of Agriculuture.

REPRESENTATIVE PUBLICATIONS:

Our research program is focused on the two major thiol-based redox buffers of the cell, thioredoxin and glutathione. These two biomolecules are largely responsible for the tightly controlled maintenance of intracellular reduction-oxidation status essential for normal cellular function. We use protein crystallography in combination with site-directed mutagenesis, enzymology, protein chemistry, and other biophysical techniques, to examine the biological functions of the enzymes responsible for maintaining reduced glutathione and thioredoxin reservoirs.

In addition to redox homeostsis, we are also involved in several collaborative projects focused on structure and function relationships in enzymes of biomedical significance. Most recently, we have been collaborating with the Simpson Lab on enzymes involved in hyaluronan metabolism.

SELECTED PUBLICATIONS:

Ekaterina I. Biterova and Joseph J. Barycki. 2009. Mechanistic details of glutathione biosynthesis revealed by crystal structures of S. cerevisiae glutamate cysteine ligase. Submitted.

Kristin Williams, Sierra Cullati, Aaron Sand, Ekaterina I. Biterova and Joseph J. Barycki. 2009. Crystal structure of acivicin-inhibited -glutamyltranspeptidase reveals a critical role for the C-terminus in autoprocessing and catalysis. Biochemistry. 48(11), 2459-2467.

Ling Zhang, Alamelu G. Bharadwaj, Andrew Casper, Joel Barkley, Joseph J. Barycki and Melanie A. Simpson. 2009. Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues. Journal of Biological Chemistry. 282(14), 9433-9442.

Amy Morrow, Kristin Williams, Aaron Sand, Gina Boanca, and Joseph J. Barycki. 2007. Characterization of H. pylori gamma-glutamyltranspeptidase reveals the molecular basis for substrate specificity and a critical role for the tyrosine 433-containing loop in catalysis. Biochemistry, 46 (46), 13407-13414.

Joseph J. Barycki. 2007. Chapter 2: Glutathione. In Redox Biology. Ed. Ruma Banerjee. Publisher: John Wiley and Sons, Inc.

Katherine E. Easley, Brandi J. Sommer, Gina Boanca, Joseph J. Barycki, and Melanie A. Simpson.  2007.  Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280.  Biochemistry, 46(2), 369-78.

Gina Boanca, Aaron Sand, Toshihiro Okada, Hideyuki Suzuki, Hidehiko Kumagai, Keiichi Fukuyama, and Joseph J. Barycki.  2007. Autoprocessing of H. pylori g-glutamyltranspeptidase leads to the formation of a threonine-threonine catalytic dyad.  Journal of Biological Chemistry, 282 (1), 534-541.

Gina Boanca, Aaron Sand, and Joseph J. Barycki.  2006. Uncoupling enzymatic and autoprocessing activities of H. pylori g-glutamyltranspeptidase.  Journal of Biological Chemistry 281 (28), 19029-19037.

Ekaterina I. Biterova, Anton A. Turanov, Vadim N. Gladyshev and Joseph J. Barycki.  2005. Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism.  Proceedings of the National Academies of Sciences 102(42), 15018-15023.

Patricia L. Herman, Mark Behrens, Sarbani Chakraborty, Brenda M. Chrastil, Joseph Barycki and Donald P. Weeks.  2005.  A three component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: Gene isolation, characterization, and heterologous expression.  Journal of Biological Chemistry 280, 24759-24767.

Peter J. Hanley, Stefan Dröse, Ulrich Brandt, Rachel A. Lareau, Abir L. Banerjee, D. K. Srivastava, Leonard J. Banaszak, Joseph J. Barycki, Paul P. Van Veldhoven, and Jurgen Daut.  2005.  5-hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for b-oxidation of fatty acids.  The Journal of Physiology 562(Pt 2), 307-318.

Brandi J. Sommer, Joseph J. Barycki, and Melanie A. Simpson.  2004.  Characterization of human UDP-glucose dehydrogenase: Cys 276 is required for the second of two successive oxidations.  Journal of Biological Chemistry 279, 23590-23596.

• Natural products biosynthesis
• Metabolic engineering
• Ceramide synthases and other sphingolipid enzymes

RESEARCH DESCRIPTION:
Biosynthetic mechanism for fungal polyketides ¾ Many important polyketide natural products are produced by filamentous fungi, such as the mycotoxin fumonisins produced by Fusarium verticillioides and the cholesterol-lowering drug lovastatin (MervacorTM) by Aspergillus terreus. We are using genetic and biochemical approaches to understand the mechanism by which the fungi synthesize these complex metabolites. The biosynthetic genes are specifically knocked out and functionally complemented to define the role of the genes. The biosynthetic enzymes are produced in heterologous hosts, such as E. coli and baker’s yeast, to determine the reactions catalyzed by these enzymes.

Genetics and function of novel antibiotics ¾ in this collaborative project, we are studying the genetics and function of a novel antifungal metabolite, HSAF, isolated from the biocontrol agent Lysobacter enzymogenes C3. MS and NMR data show that HSAF has a chemical structure that is distinct from any known fungicides or antifungal drugs. Genetic analysis of a model fungus has showed that HSAF targets the biosynthesis of a specific group of sphingolipids that are required for polarized hyphal growth. HSAF represents a promising candidate for new fungicides and antifungal drugs with new chemistry and an unprecedented mode of action. A gene cluster has been cloned and sequenced. Among them, a PKS gene has been knocked out, and the resulted mutants lost the ability to produce HSAF and to inhibit fungal growth. The results demonstrate the polyketide origin of HSAF biosynthesis. The biosynthetic genes provide the basis for genetic engineering to produce new fungicides and antifungal drugs.

RESEARCH INTERESTS:

• Molecular mechanisms involved in the regulation of cell death by redox signaling
• Molecular mechanisms involved in the regulation of cellular transformation by redox signaling
• Cell death and Neurodegenerative diseases
• Environmental Toxicity and Oxidative stress
• Molecular Mechanisms of Cytotoxic Brain Edema
  

RESEARCH DESCRIPTION:

Cell Death and Redox Signaling

Cell death is a central phenomenon in the etiology of several human diseases including cancer and neurodegenerative disorders. It has been demonstrated that alterations in the cellular redox balance regulate the activation of distinct signaling cascades leading to the progression of a variety of cell death programs. However, the exact mechanisms involved in the regulation of cell death by redox signaling are still far from being understood.

In our research group we aim to elucidate the molecular mechanisms involved in the regulation of cell death progression by redox signaling. Because the intracellular thiol-disulfide (GSH/GSSG) balance is considered the major determinant of the redox status of the cell, we are particularly interested in studying how alterations in thiol homeostasis regulate the activation/inactivation of the cell death machinery during the pathogenesis of cancer and neurodegeneration.

Environmental Toxicity and Oxidative Stress

The environment represents a key contributor to human health and disease. Exposure to many toxicants such as metals and pesticides have detrimental effects on health and are considered to contribute substantially to a number of diseases of major public health significance. It has been recognized that many of the toxic effects induced by environmental stressors are mediated by regulation/induction of cell death and oxidative stress whose deregulation has been associated to several environmental diseases. The overall impact of environmental changes on the mechanisms of cell death progression is poorly understood yet the consequences of modifying/regulating them can result in a potential increased risk of developing diseases such as cancer and neurodegeneration, which are associated to alterations in cell death rates. We are interested in identifying the molecular mechanisms by which oxidative stress regulates cell death during environmental toxicity.

Cytotoxic Brain Edema

Cytotoxic swelling is an important component of brain edema which occurs during distinct pathological states including hyponatraemia, traumatic brain injury, ischemia and hepatic encephalopathy. It is the result of a deregulated cell swelling of neurons and glia and the subsequent reduction in the extracellular space. Cytotoxic edema results mainly from both extracellular and/or intracellular osmotic disturbances and is also associated to the impairment of volume regulatory mechanisms. In our research group, we are also studying the signaling cascades involved in the regulation of cytotoxic swelling. We are particularly interested in characterizing the signaling events that modulate the activation of ionic/osmolyte efflux pathways under these circumstances.

REPRESENTATIVE PUBLICATIONS:

• Pasantes-Morales H, Franco R, Ordaz B, Ochoa LD. Mechanisms counteracting swelling in brain cells during hyponatremia. Arch Med Res. 2002; 33(3):237-44.
• Pasantes-Morales H, Franco R. Astrocyte Cellular Swelling: Mechanisms and Relevance to Brain Edema. In: The Role of Glia in Neurotoxicity. (Aschner M., Ed) 2005. CRC Press. Chapter 10 pp. 173 - 190
• Franco R, Bortner CD, Cidlowski JA. Potential roles of electrogenic ion transport and plasma membrane depolarization in apoptosis. J Membr Biol. 2006; 209(1):43-58.
• Franco R, Cidlowski JA. SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: Glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis. J Biol Chem. 2006; 281(40):29542-29557.
• Franco R, Schoneveld, OJ, Pappa A, Panayiotidis MI.  The central role of glutathione in the pathophysiology of human diseases. Arch Physiol Biochem. 2007; 113(4):234-58. (corresponding author)
• Franco R, Panayiotidis MI and Cidlowski JA. Glutathione depletion is necessary for apoptosis in lymphoid cells independent of Reactive Oxygen Species Formation. J Biol Chem. 2007; 282(42):30452-30465
• Pappa A, Franco R, Schoneveld O, Panayiotidis MI. The biochemical basis of sulfur protection against oxidant-mediated lung disease. Curr Med Chem. 2007; 14(24):2590-2596.
• Franco R, Panayiotidis MI, Ochoa-De la Paz LD. Autocrine regulation of cell volume: the role of plasma membrane receptors and released transmitters. J Cell Physiol. 2008;216(1):14-28. (corresponding author)
• Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI. Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett. 2008; 266(1):6-11
• Franco R, DeHaven WD, Sifre M, Bortner CD, Cidlowski JA. Glutathione transport regulates cell shrinkage and potassium loss in FasL-induced apoptosis in Jurkat cells. J Biol Chem. 2008; 283(52):36071--36087
• Franco R, Panayiotidis MI. Environmental toxicity, oxidative stress, human disease and the “black box” of their synergism: How much have we revealed? Mutat Res. 2009; 674(1-2): 1-2
• Franco R, Sanchez-Olea R, Reyes-Reyes EM, Panayiotidis MI. Environmental toxicity, oxidative stress and apoptosis: Ménage à Trois. Mutat Res. 2009; 674(1-2): 3-22 (corresponding author)
• Franco R, Cidlowski JA. Apoptosis & Glutathione: Beyond an antioxidant. Cell Death Differ. 2009;16(10):1303-14
  

RESEARCH DESCRIPTION:
In the past two decades of research activities bold and sustained contributions were made towards understanding how alterations in mononuclear phagocyte function induce metabolic changes in the brain and ultimately lead to neural cell damage during AIDS dementia. Similar mechanisms were found operative in other neurodegenerative disorders making the discoveries broadly applicable to neurological disease. Importantly, such discoveries have significant implications towards finding effective therapies or preventative strategies for many neurodegenerative disorders; including, but not limited to, Alzheimer's and PD. Indeed, neuroprotective and neuroregenerative strategies for neurodegenerative disorders are now underway utilizing a breadth of novel approaches in basic and applied immunology.

• Thiol-based redox regulation and signaling
• Identity and functions of selenocysteine-containing proteins
• Mechanism of cancer prevention by selenium
• Bioinformatics
• Redox biology
• Aging
   
  

SELECTED PUBLICATIONS:

• Kryukov, G. V., Castellano, S., Novoselov, S. V., Lobanov, A. V., Zehtab, O., Guigo, R., and Gladyshev, V. N. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439-4313.

• Koc, A., Gasch, A. P. Rutherford, J. C., Kim, H.-Y., and Gladyshev, V. N. (2004) Methionine sulfoxide reductase regulation of yeast lifespan reveals ROS-dependent and ROS-independent components of aging. Proc. Natl. Acad. Sci. USA 101, 7999-8004.

• Kim, H. Y., and Gladyshev, V. N. (2005) Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases. PLoS Biol. 3, e375.

• Xu, X.-M., Carlson, B. A., Mix, H., Zhang, Y., Saira, K., Glass, R. S., Berry, M. J., Gladyshev, V. N., and Hatfield, D. L. (2007) Biosynthesis of selenocysteine on its tRNA in eukaryotes. PLoS Biol. 5, e4.

• Fomenko, D. E., Xing, W., Adair, B. M., Thomas, D. J., and Gladyshev, V. N. (2007) High-throughput identification of catalytic redox-active cysteine residues. Science 135, 387-389.

RESEARCH DESCRIPTION:
Dr. Lin’s lab devotes to translational researches, briefly as follows:

1) To investigate the molecular mechanism by which human PCa cells can grow in the androgen-reduced environment. Based on studies examining the functional role of cellular prostatic acid phosphatase (cPAcP), Dr. Lin’s lab has discovered how cPAcP is involved in regulating androgen-stimulated cell proliferation. Dr. Lin can restore androgen-sensitivity in androgen-independent PCa cells by the expression of cPAcP. They also found that this enzyme directly suppresses prostate tumor growth, which is validated by the knockout experiment in animals. These findings could lead to the development of new strategies to control prostate carcinogenesis and new treatments for PCa. The application of cPAcP as a potential therapeutic agent has been awarded a US Patent (August 2006).

To define the biological activity of acid phosphatases, Dr. Lin’s group proposes the “Re-visiting of Histidine-dependent acid phosphatase as a distinct sub-group of tyrosine phosphatases”. Several lines of evidence support the hypothesis that this group of enzymes plays a critical role in regulating cell growth and differentiation by controlling tyrosine phosphorylation in cells from prokaryotes to human cells. This novel concept will make a significant impact on the field of research in tyrosine phosphatases.

To prevent PCa, Dr. Lin’s lab reveals that redox signaling plays a critical role in mediating “non-genomic” androgen action on cell growth. The data further reveal a signal pathway from steroid hormone androgen via redox to tyrosine phosphorylation signal transduction, leading to cell proliferation. This seminal discovery provides with a mechanistic explanation to prostate carcinogenesis induced by androgens, leading to cancer prevention.

2) To improve the treatment for advanced PCa patients. Dr. Lin’s lab is investigating novel strategies, including new applications of drugs, pro-drug development and nanomedicine approach. For example, Dr. Lin previously discovered that mifepristone, i.e., RU486, can suppress hormone-refractory PCa growth in xenograft animals. Anecdotal clinical results were observed and clinical trials are going on now.

He currently hypothesizes that combinational treatments of androgen-independent PCa cells with chemotherapeutic reagents plus inhibitors to signaling transduction pathways have enhanced efficacies on inducing apoptosis of those cells. Other avenues include testing new compounds, formulating nanomedicine and developing novel reagents for expressing genes specifically in PCa cells. These reagents together can be used potentially to formulate specific, effective therapy protocols toward PCa.

3) To identify new markers/targets for developing novel therapies. In advanced prostate carcinomas, neuroendocrine (NE) cells, the third cell population in normal prostate, rise. It is proposed that NE cells secret stimulating factors, which in turn promote the growth and progression of PCa cells under androgen-reduced environment. Dr. Lin’s lab has established NE cell lines derived from PCa cells in culture. These NE cell lines which were awarded with two US patents (August 2004 & July 2007) will be useful for developing novel strategies of treating advanced hormone-refractory PCa patients.

To develop system biology, i.e., identifying new markers for diagnosis and novel targets for therapy, for future personized medicine, Dr. Lin’s lab utilizes the cDNA microarray and microRNA technologies and proteomic approaches. Currently, they are characterizing those newly identified molecules serving as key biomarkers in cancer diagnosis as well as prognosis and/or as novel targets for treatments.

Facilities:  Spectroscopy and Biophysics Core.

The role of the Spectroscopy and Biophysics Core is to provide instrumentation, training and support for any type of experimental work involving biophysical and spectroscopic measurements.  Although most of the instruments listed below are configured for use in protein characterization work, the services that our core provides can be extended to partially purified proteins, whole cell assays and analysis of tissues.  The instrumentation is located in rooms E155 and N113B of the Beadle Center and includes an Inductively Coupled Plasma Mass Spectrometer (Agilent 7500 cx), an HPLC with diode array detector (Agilent LC1200), a 96-well plate autosampler (Elemental Scientific Inc), a Stopped Flow rapid kinetics instrument (Hi Tech), a circular dichroism with rapid scanning capability (Olis, Inc.), a differential scanning calorimeter (Microcal GE Healthcare), a isothermal calorimeter (Microcal GE Healthcare) and a Spectrofluorimeter (Varian, Cary Eclipse).

Services Offered by the RBC Spectroscopy Facility:

• Determination of protein stability and thermodynamic parameters for protein conformational changes by means of differential scanning calorimetry (DSC).  During a DSC experiment, the temperature of the sample cell and a reference cell is ramped up - usually in a linear fashion, and the difference in heat that is necessary reach that temperature is continuously monitored.  The heat measurement is translated into enthalpies of transitions and heat capacities.
• Ligand-protein and protein-protein binding studies by means of isothermal microcalorimetry.  A titrator syringe adds a concentrated solution to a sample of protein.  The heat evolved or taken during the binding process or a chemical reaction is monitored at a constant temperature set by the user.  After correction for heat of dilution of the titrant the enthalpy of binding can be accurately estimated.  Studies at several temperatures allow the measurement of entropies and free energies ligand-macromolecule interactions and macromolecule-macromolecule interactions such and protein-protein and protein-DNA complexes.
• Measurement of secondary structure and secondary structure changes by circular dichroism.  Experiments can be carried out in the UV and visible range and changes as small as 0.1 mA or ellipticity can be obtained both in spectral and kinetic modes.  The instrument can also be modified for measurement of fluorescence polarization measurements, rapid scanning fluorescence and rapid scanning absorbance measurements
• Stopped flow spectrophotometry.  Rapid mixing can be achieved in 0.2 milliseconds.  Measurements in absorbance, fluorescence and chemiluminescence are routinely performed with detection in single wavelength and diode array (512 nm per scan) configurations.  Accessories included are double mixing experiments, a chemical quench 6 port valve (Q-pod) and nitrogen gas attachments for anaerobic experiments
• Standard Spectrofluorimeter equipped with up to 6 cuvets, a 96-well plate holder and a Peltier thermostated single cuvet holder for fluorescence, phosphorescence and chemiluminescence experiments in spectral and kinetic modes.
• Elemental Analysis by means of Inductively Coupled Plasma Mass Spectrometry.  Almost any element of the periodic chart can be determined down to the parts per trillion (ng per kg or picomolar) concentration except C,H, O, N, F, Cl and noble gases.  Samples can be as small as 100 microliters and can be in either liquid or solid states.  An HPLC unit is coupled with this instrument which allow the separation of elements in distinct chemical environments (chemical speciation) or the reduction of interferences by reverse phase or ion chromatography.  In addition, isotope ratio and spectral semi-quantitative analysis can be performed.
• Analytical Ultracentrifugation.  Determination of the protein quaternary structure of proteins using equilibrium analytical ultracentrifugation of protein solutions in water and deuterium oxide.

Summary of Equipment in the Spectroscopy Core Facility:

Agilent ICP-MS 7500cx with the autosampler shown in the background.

ESI autosampler can handle up to 576 samples per run.

Cary Eclipse Spectrofluorimeter with 96-well plate holder in place.

Hi-Tech Stopped flow showing the sample handling unit, monochromator, photomultiplier power supply and diode array detector.

Microcal Differential Scanning Calorimeter and ancillary computer.

Microcal Isothermal Calorimeter and computer.  The syringe titrator is not shown for clarity.

Olis-RSM 1000 setup as a rapid scanning spectrophotometer.

• 1998: University of Guelph, Ontario, Canada (PhD)
• 1998-2000: Institut Pasteur, Paris, France (postdoctoral training)
• 2000-2003: Brigham and Women’s Hospital, Harvard Medical School, Boston, MA (postdoctoral training)
• 2003-2006: Brigham and Women’s Hospital/Harvard University, Boston, MA (instructor)
   

RESEARCH INTERESTS:

Our long-term interest is the cellular and molecular mechanisms underlying the development of human autoimmune diseases.  Most healthy humans have a propensity to develop autoimmune diseases, as evidenced by the presence of autoreactive T cells or autoantibodies in their naïve periphery.  T cells are educated in the thymus, where high-affinity TCR-bearing cells that interact with self-antigens are deleted by negative selection while T cells with low-affinity TCR to self-antigens are selected positively and exported to the periphery. In spite of the high frequency of self-reactive T cells in the naïve peripheral repertoires, autoimmune responses do not ensue spontaneously.  How this tolerance is maintained is a fundamental question.  We focus our investigations on the role of reactive oxygen species in the maintenance of T cell tolerance and autoimmunity.

REPRESENTATIVE PUBLICATIONS:

Reddy J, Estelle Bettelli, Lindsay Nicholson, Mei-Huei Jang, Kai W. Wucherpfennig, and Vijay K. Kuchroo.  Detection of autoreactive myelin proteolipid Protein 139-151-specific T cells by using MHC II IAs tetramers. J Immunol, 2003, 170: 870-877.

Illes Z, Stern JN, Reddy J, Waldner H, Mycko MP, Brosnan CF, Ellmerich S, Altmann DM, Santambrogio L, Strominger JL and Kuchroo VK. Modified amino acid copolymers suppress myelin basic protein 85-99-induced encephalomyelitis in humanized mice through different effects on T cells. Proc. Natl. Acad. Sci. U S A. 2004, 101:11749-54.

Stern JN, Illes Z, Reddy J, Keskin DB, Sheu E, Fridkis-Hareli M, Nishimura H, Brosnan CF, Santambrogio L, Kuchroo VK and Strominger JL.  Amelioration of proteolipid protein 139-151-induced encephalomyelitis in SJL mice by modified amino acid copolymers and their mechanisms. Proc. Natl. Acad. Sci. U S A. 2004, 101(32): 11743-8.

Anderson AC, Reddy J (co-first authors), Nazareno R, Sobel RA, Nicholson LB and Kuchroo VK. IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice.  J. Immunol.  2004, 173: 828-834.

Greve B, Reddy J, Waldner HP, Sobel RA and Kuchroo VK. Dissimilar background genes control susceptibility to autoimmune disease in the context of different MHC haplotypes: NOD.H-2(s) congenic mice are relatively resistant to both experimental autoimmune encephalomyelitis and type I diabetes.  Eur. J. Immunol. 2004, 34:1828-1838.

Reddy J, Illes Z, Zhang X, Encinas J, Pyrdol J, Nicholson L, Sobel RA, Wucherpfennig KW and Kuchroo VK.  Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis.  Proc. Natl. Acad. Sci. U S A. 2004, 101(43): 15434-9.

Stern JN, Illes Z, Reddy J (co-first authors), Keskin DB, Sheu E, Fridkis-Hareli M, Kuchroo VK and Strominger JL.  Peptide 15-mers of defined sequence that substitute for random amino acid copolymers in amelioration of experimental autoimmune encephalomyelitis.  Proc. Natl. Acad. Sci. U S A. 2005, 102(5): 1620-5.

Dardalhon V, Schubart, A, Reddy J, Meyers J, Monney L, Sabatos C, Ahuja R, Nguyen K, Freeman G, Greenfield E, Sobel R and Kuchroo V. CD226 is specifically expressed on the surface of Th1 cells and regulates their expansion and effector functions. J. Immunol. 2005, 175: 1558-1565.

Reddy J, Waldner H, Zhang X, Illes Z, Wucherpfennig KW, Sobel RA, and Kuchroo VK. CD4+CD25+ regulatory T cells contribute to gender differences in susceptibility to experimental autoimmune encephalomyelitis.  J. Immunol (cutting edge): 2005, 175: 5591-5595.

Xiao S, Najafian N, Reddy J, Albin M, Zhu C, Jensen E, Korn T, Anderson AC, Zhang Z, Gutierrez C, Moll T, Sobel R, Umetsu D, Yagita H, Akiba H, Sayegh M, DeKruyff R, Khoury S and Kuchroo V. Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function. 2007. J. Exp. Med. 204: 1691-702.

Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen T, Bäckström B, Wucherpfennig K, Strom T, Oukka M and Kuchroo V. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.  2007. Nat. Med. 4: 423-31.

RESEARCH DESCRIPTION:
Environmental modulation of PIA synthesis. Staphylococcal medical device-associated infections usually involve a two-step process leading to the formation of a bacterial biofilm. The first step in biofilm formation involves attachment of the organism to an uncoated plastic surface, or a plastic surface coated with host proteins. The second step involves the accumulation of bacteria on top of the bacteria adhering to the plastic surface, a step requiring the production of polysaccharide intercellular adhesin (PIA). PIA synthesis  is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same nutritional and stress conditions repress tricarboxylic acid (TCA) cycle activity; leading us to hypothesize that altering TCA cycle activity would affect PIA production. Confirmation of this hypothesis is based in part, on our observation that culturing S. epidermidis with a low concentration of a TCA cycle inhibitor (fluorocitric acid) dramatically increases PIA synthesis. Taken together, these data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, and/or the redox status of the bacteria. Currently, we are measuring the changes in intracellular metabolite pools that accompany the induction of PIA synthesis with the goal of identifying regulators capable of responding to these changes.

Posttranscriptional regulation of staphylococcal virulence. Staphylococcal aconitase is homologous (52% amino acid identity) to the bifunctional iron-responsive protein-1 (IRP-1). IRP-1 is a eukaryotic mRNA-binding protein that posttranscriptionally regulates the synthesis of iron-regulated proteins, and has aconitase enzymatic activity. Like IRP-1, aconitase from Bacillus subtilis and Escherichia coli can bind to and posttranscriptionally regulate mRNAs (Alen and Sonenshein 1999. Proc. Natl. Acad. Sci. 96:10412-10417; Tang and Guest 1999. Microbiology 145:3069-3079). These observations established bacterial aconitase, like IRP-1, as a bifunctional protein. In S. aureus, aconitase inactivation decreases production of virulence factors and enhances long-term survival relative to an isogenic strain. Some of the deleterious effects of aconitase inactivation are the result of the loss of enzymatic function (i.e., a metabolic block of the TCA cycle) and some are the result of the loss of regulatory function. We are conducting research to identify which effects of aconitase inactivation are the result of a metabolic defect and which are due to the loss of aconitase regulatory function.
           

RESEARCH INTERESTS:

• Structural biology of Parkinsonism-associated proteins
• Structural and functional studies of the DJ-1 superfamily
• Atomic and ultra-high resolution X-ray crystallography
  

RESEARCH DESCRIPTION:

We are interested in understanding the biochemical basis of Parkinson’s disease (PD) by using a combination of structural and biophysical approaches to study certain proteins involved in the disease.  PD is a progressive neurodegenerative disease that results from the death of dopaminergic neurons in the midbrain. The causes of the common sporadic form of PD are unknown; however dramatic progress in the understanding of the biochemical foundations of Parkinsonism has been made through the study of rare, heritable forms of the disease.  Genetic studies of these heritable forms of PD have led to the identification of several genes whose mutation causes Parkinsonism, and we are structurally and biophysically characterizing these proteins to achieve an atomic level of understanding of their functions.

• Mitochondrial-produced reactive oxygen species
• Hypertension
• Neural control of cardiovascular function
• Amyotrophic lateral sclerosis (Lou Gehrig’s Disease)

Research in the laboratory is focused on the role of mitochondrial-produced reactive oxygen species (ROS) in diseases associated with the central nervous system (CNS). We primarily study two different CNS-associated diseases: 1) Angiotensin II (AngII)-dependent neurogenic hypertension; 2) Amyotrophic lateral sclerosis (ALS, also known at Lou Gehrig’s Disease).

Increased circulating levels of AngII can lead to the development of neurogenic hypertension by acting on specialized brain regions known as circumventricular organs, which lack a blood-brain-barrier, and altering central cardiovascular outputs including sympathoexcitation. To better understand the central actions of AngII in the development of neurogenic hypertension and to identify novel, central therapeutic targets of the disease, our lab investigates the signaling mechanisms of AngII in the CNS. Previously, ROS generated by NADPH oxidase have been identified as important signaling intermediates in central AngII-mediated cardiovascular effects. However, additional sources of ROS including mitochondria, which are the primary sites for ROS generation in most cells, have yet to be investigated. Utilizing adenovirus-mediated gene transfer of mitochondrial-targeted antioxidants both in vitro and in vivo, our lab hopes to provide new insight into the role of mitochondrial-produced ROS in central AngII signaling.

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. One of the most significant findings to date in ALS research has been the identification of mutations in the gene encoding CuZnSOD, in a subset of familial ALS patients. Mitochondria have become increasingly suspicious as key players in the development and progression of ALS; however, the precise role of mitochondria in the pathogenesis of ALS remains unclear. We have previously shown that mutant CuZnSOD increases ROS levels in mitochondria and that overexpression of the mitochondrial-targeted antioxidant manganese superoxide dismutase (MnSOD) attenuates mutant CuZnSOD-mediated neuronal toxicity. Our lab will continue to study the role of mitochondrial-produced ROS in ALS by modulating levels of mitochondrial-targeted antioxidants in both a cell culture model and an ALS transgenic mouse model.