The mission of the Uniformed Services University of the Health Sciences is to educate, train and prepare uniformed services health professionals, officers and leaders to directly support the Military Health System, the National Security and National Defense Strategies of the United States and the readiness of our Armed Forces.
Since our first graduating class in 1982, the USU's MDs. Nurses and graduates in biomedical sciences provide exceptional service through service in the U.S. Military and civilian careers of distinction. Today, America's Medical School has 691 enrolled students and 5,043 graduates. Over 1,300 graduates in Biomedical Sciences lead aggressive research in medical research. Today's 663 graduates of the School of Nursing blend science, research and field training in advanced practice and PhD degrees. The USU's Postgraduate Dental College provides advanced degree's to the military's dental community, graduating 72 students since establishment.
The University's research program covers a range of clinical and other topics important to both the military and public health. Infectious diseases, trauma medicine, health maintenance, and cancer are areas of particular strength. Researchers are also making important new efforts in state-of-the-art fields that cut across disciplines, such as genomics, proteomics, and drug-delivery mechanisms.
USU is home to many different Centers and Institutes, which help advance the university's research, education and public service missions. Faculty members and students collaborate with other leading experts at USU's Centers and Institutes on projects that push incredible boundaries across manifold disciplines of biomedical science. Their work is shaping military medicine and world health in many positive, powerful ways.
The USU's military unique curriculum is supported by military professions from all services who teach USU's military and civilian students. All military personnel are supported by the USU Brigade, the Brigade staff are managed by the Military Personnel Office.
AFRRI mission is to preserve the health and performance of U.S. military personnel and to protect humankind through research that advances understanding of the effects of ionizing radiation.
To these ends, the institute collaboratively researches the biological effects of ionizing radiation and provides medical training and emergency response to manage incidents related to radiation exposure.
Chronic diseases and even aging itself are known to damage the body by dys-regulated inflammatory processes. Dysregulated expression of the pro-inflammatory cytokine and chemokine genes including interleukin-8 (IL-8) are known to contribute to chronic inflammatory diseases. The expressions of such genes are known to be regulated by post-transcriptional mechanisms. Recently, microRNAs (miRNAs) have been proven to be key post-transcriptional regulators of gene expression by directing their target mRNAs towards degradation and/or translational repression. The mis-regulation of specific miRNAs has been demonstrated in a variety of diseases in humans including cancer, heart disease and diabetes. Since each miRNA governs the expression of multiple genes, the most effective way to overcome the effects of a mis-regulated miRNA is to modulate its expression in diseased cells. Thus, miRNAs have emerged as important therapeutic targets in the frontier of biomedical research. Recent studies indicate that even a single miRNA can induce a therapeutic response in an animal model of disease. My laboratory is currently focused on studying mechanisms specific for the pro-inflammatory disease phenotype in Cystic Fibrosis as well as general mechanisms that cause dys-regulation of inflammation, a phenomenon characteristic of a milieu of diseases including cancer, cardiovascular diseases, and immune system disorders. My laboratory is involved in investigating such mechanisms.
Mechanism of Regulation of inflammation in Cystic Fibrosis: Cystic Fibrosis (CF) is the most common life limiting recessive disease in the U.S., and is due to mutations in the CFTR gene. CF mutations, of which the most common is F508-CFTR, cause a massive pro-inflammatory phenotype in the lung, which is characterized by high levels of interleukin-8 (IL-8). The disease phenotype seems to be intrinsic to the CF condition, since fetal CF lung epithelium secretes massive levels of IL-8, in vivo, even in the absence of detectable infection. The problem is that the mechanism by which IL-8 gene expression is dysregulated in CF is not known. However, we have shown that CF lung epithelial cells in culture not only secrete large amounts of IL-8 protein, but also have high levels of very stable IL-8 mRNA. Thus, for a reason yet to be elucidated, IL-8 mRNA is degraded very slowly in CF lung epithelia. On this basis, our approach has been to investigate the mechanism by which IL-8 mRNA is rendered aberrantly stable in CF lung epithelial cells. The rationale is that understanding this dysfunctional regulatory mechanism may lead to more focused anti-inflammatory strategies for CF therapy.
Recently, a novel class of endogenous non-coding RNA molecules known as microRNAs (miRNAs) has emerged as important targets in the frontier of biomedical research. These small ~22 nucleotides long RNAs have been proven to be key regulators of gene expression by directing their target mRNAs towards degradation and/or translational repression. The mis-regulation of specific miRNAs has been demonstrated in a variety of diseases in humans including cancer, heart disease and diabetes. CF lung epithelial cells in culture exhibit mis-expression of specific miRNAs, including miR-155, compared to control cells, both in culture and in ex vivo bronchial biopsies of CF patients. The failure of CFTR channel activity, either by chemical inhibition or by mutation, results in aberrantly enhanced expression of miR-155.
Regulation of microRNA biogenesis and function in inflammation: The goal of this project is to determine the mechanism by which biogenesis and function of miR-155 is regulated by inflammation and how miR-155 is a potent inducer of inflammation. The function and expression of mature miRNAs is controlled by mechanisms that regulate the processing of primary (pri-) and precursor (pre-) miRNAs. Our objective is to investigate the mechanisms of alterations in processing of miR-155 and how that leads to its aberrant expression and dysfunction in inflammatory response. We are using two model systems; pro-inflammatory CF lung epithelial cells characterized by hyper-expression of the pro-inflammatory chemokine interleukin-8 (IL-8), as well as macrophage cells stimulated with inflammatory stimuli. We have found significant elevated expression of miR-155 in CF cells compared to controls and uncovered enhanced processing of the miR-155 precursor as the basis for this increase. Roberto Gherzi's laboratory (Italy) has shown increased expression of miR-155 in LPS-stimulated macrophages. Furthermore, we found that CF cells express negligible endogenous levels of the inflammation-associated RNA-binding protein (RBP) Tristetraprolin (TTP), which accelerates mRNA degradation through recognition of adenine and uridine (AU)-rich sequences. Moreover, over-expression of TTP not only suppresses IL-8 expression through destabilization of IL-8 mRNA but also inhibits miR-155 processing. It has also been shown that KH-type splicing regulatory protein (KSRP), another AU-interacting RBP associated with inflammation, also enhances miR-155 processing in CF cells. Therefore, expression and function of miR-155 is likely to be regulated by the inflammatory RBPs, TTP and KSRP, and that reciprocally miR-155 is also a potent inducer of inflammatory genes. We are in the processing of determining (i) the mechanism by which KSRP enhances miR-155 processing, (ii) how the AU-binding protein TTP regulates miR-155 expression, and (iii) how miR-155 regulates the pro-inflammatory IL-8 gene expression.
Utilizing an innovative combination of in vitro techniques for mapping RNA secondary structures, cell culture-based assays, immunoprecipitation as well as in vivo affinity purification techniques, this project is targeted towards providing new insights into miRNA biogenesis and highlight how variations in these maturation pathways might underlie select human diseases.
Postdoctoral fellowship, Carnegie Institution, Department of Embryology, Baltimore, MD
Ph.D., Curriculum in Genetics and Molecular Biology, University of North Carolina - Chapel Hill, Chapel Hill, NC
B.A., University of Pennsylvania, Philadelphia, PA
Mitochondria are cellular organelles that produce the majority of ATP in eukaryotic cells. Historically, they have been intensively studied biochemically, and many of the metabolic pathways that take place in mitochondria, such as the TCA cycle and electron-transfer, are well characterized. Mitochondria are unique in that they contain their own DNA, mtDNA. While this DNA is small, approximately 16kb in metazoans, it is critical for normal mitochondrial function. Because mitochondria cannot be made de novo, all of your mitochondria and mtDNA were inherited from your mother's oocyte cytoplasm.
In the last twenty years, an increasing number of diseases have been linked to mutations in either mtDNA or nuclear genes encoding mitochondrial proteins. While the general observation that faulty mitochondria can lead to disease may not be surprising given the important role mitochondria play in cellular function, the specificity with which only certain cell types are affected by single mutations has been. In addition, there is mounting evidence for a role of mitochondrial dysfunction in common diseases, such as neurodegenerative disease and diabetes.
In the past decade or so, a previously under-appreciated aspect of mitochondrial biology has come to light. Mitochondria are not simply static ATP-producing machines (the so-called "power house of the cell"), but are instead very dynamic. They change shape and size due to growth and fission/fusion, and mitochondria can rapidly transit around the cell by moving along the actin and microtubule cytoskeleton.
The broad interests in my lab are studying how mitochondria change shape, location physiology and mtDNA content in response to developmental changes, and elucidating which genes and pathways are responsible. To address these general questions, we use the model system Drosophila melanogaster, or fruit fly. The advantages to using fruit flies are that they have a rich genetic history, allowing rapid and straightforward mutant acquisition, researchers understand much about organ and tissue development, and mitochondria can be imaged in fixed and live tissue at single organelle resolution. While fruit flies are interesting in and of themselves, it is important to note that an estimated 75% of human disease genes have a functional homolog in flies. The overwhelming similarity of genes and molecular pathways between humans and flies allows researchers to apply knowledge gained from Drosophila to elucidate the causes of human disease.
Understanding mitochondrial inheritance
mtDNA has a higher mutation rate than nuclear DNA and mitochondria are maternally inherited. Despite this, females can reproducibly and reliably produce hardy offspring. We are interested in identifying the genes and mechanisms involved in ensuring the mother deposits only highly functional mitochondria into the oocyte in order to support embryonic development. We can clearly visualize mitochondria during all of oogenesis in both fixed and live tissue and we have characterized several genes involved in normal mitochondrial function in the ovary. Mitochondria exhibit stereotypical changes during oogenesis (for one example, see image below), thus mutations that perturb mitochondrial localization or function can be readily identified.
The gene we are currently studying, clueless, is involved in mitochondrial localization and function. Flies mutant for clu are sterile, uncoordinated and have reduced life-spans. In clu mutant ovaries, mitochondria are distinctly mislocalized to the plus-end of microtubules. Clu protein is highly conserved and present in particles in the germline.
We believe clu acts to maintain mitochondrial function. When absent, mitochondria accumulate damage and subsequently undergo directed movement to the plus-ends of microtubules.
Human MultidrugTransporter: Mode of Action and Functional Regulation.
The effectiveness of anti-microbial and anti-cancer chemotherapy largely depends on the ability of thetherapeutic agents to reach their sites of action. Following administration, the fate of a drug molecule depends on how well it is absorbed from its site of administration, its distribution pattern, the extent and nature of its biotransformation, and on the efficiency by which it is excreted. Even when these obstacles are surpassed, thetherapeutic potency of a drug could be profoundly affected by occurrence of intrinsic as well as acquired drug resistance in the target cells. Thus, strategic development of chemotherapeutic drugs has to continuously battle against poor bioavailability and occurrence of drug resistance. The role of the human multidrug transporter P-glycoprotein (Pgp) in both of these phenomena is rapidly unfolding. Functionally, Pgp is an ATP-dependent efflux pump for an inordinately widerange of structurally unrelated hydrophobic drugs including anti-cancer and anti-HIV agents. In order to retain thetherapeutic effectiveness of chemotherapeutic agents, a major effort is underway to selectively inhibit the function of Pgp in tumor cells as well as in certain normal tissues. Although random screening of natural products and synthetic libraries have shown some promise, a better understanding of the mechanism of Pgp-mediated drug transport is necessary for developing inhibitors with improved efficacy.
Research goals of our laboratory are directed towards 1) elucidation of the molecular mechanism involved in coupling of ATP hydrolysis to drug translocation by Pgp, 2) characterization of its functional regulation by pharmacological agents as well as endogenous molecules and 3) identification of novel therapeutic targets within the protein. We use a baculovirus mediated heterologous expression system for generation and biochemical characterization of recombinant Pgp molecules. Stable and transient transfectants of mammalian cell lines are used for assessing transport properties of the recombinant proteins.
Fong, W. F., Wan, C. K., Zhu, G. Y., Chattopadhyay, A., Dey S., Zhao, Z., and Shen, X. L.. (2007) Schisandrol A from Schisandra chnensis reverses P-glycoprotein-mediated multidrug resistance by affecting Pgp-substrate complexes. Planta Medica, 73: 212-220.
Wan, C. K., Zhu, G. Y., Shen, X. L., Chattopadhyay, A., Dey, S., and Fong, W. F. (2006) Gomisin A alters substrate interaction and reverses P-glycoprotein mediated multidrug resistance in HepG2-DR cells. Biochemical Pharmacology, 72(7): 824-837.
Maki, N., and Dey, S. (2006) Biochemical and pharmacological properties of an allosteric modulator site of human P-glycoprotein. Biochemical Pharmacology 72:145-155 Maki, N., Moitra, K., Ghosh, P., and Dey, S. (2006) Allosteric modulation bypasses requirement for ATP hydrolysis in regenerating low-affinity transition state conformation of human P-glycoprotein. Journal of Biological Chemistry . 281(16): 10699-10777
Ghosh, P., Moitra, K., Maki, N., and Dey, S. (2006) Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates. Archives of Biochemistry and Biophysics . 450 (1): 100-112.
Maki, N., Moitra, K., Silver, C., Ghosh, P., Chattopadhyay, A. K., and Dey, S. (2006) Modulator induced interference in functional cross talk between the substrate- and the ATP- sites of human P-glycoprotein. Biochemistry 45: 2739-2751
Maki, N., Hafkemeyer, P., and Dey, S. (2003). Allosteric modulation of human P-glycoprotein: Inhibition of transport by preventing substrate translocation and dissociation. Journal of Biological Chemistry. 278(20):18132-18139.
Dey, S. (2002) Biricodar (Vertex Pharmaceuticals). Current Opinion in Investigational Drugs, Vol. 3(5): 818-823.
The research interests in my lab are focused on identifying and characterizing the enzymes responsible for sphingolipid synthesis, on determining how sphingolipid synthesis is regulated, and on elucidating the physiological functions of these important lipids.A combined genetic and biochemical approach using the model eukaryote, Saccharomycescerevisiae is being used.The sphingolipids in Saccharomyces cerevisiae contain a mannosyldiinositolphosphoryl head group attached to a ceramide.The ceramide is comprised of an a-hydroxy-C26-fatty acid and phytosphingosine.We discovered that deletion of either the CSG1 or CSG2 gene results in inability to efficiently mannosylate inositolphosphorylceramide and consequently in the overaccumulation of inositolphosphorylceramide (1-3).Although these mutants grow normally under most conditions, they are exquisitely sensitive to Ca2+, and the sensitivity to Ca2+ is correlated with the overaccumulation of inositolphosphorylceramide due to the block in mannosylation.Based on this observation, we devised screens for second-site suppressor mutants that reverse the Ca2+ sensitivity of the csg2 mutant.Because many of the suppressor mutations reduce the synthesis of inositolphosphorylceramide, these genetic screens have been extremely powerful at identifying sphingolipid biosynthetic genes.Initially we screened for suppressors at 37oC and generated the SCS (Suppressor of Ca Sensitivity) collection (3-6).However, as our understanding of the suppressor mutants evolved, we realized there would be advantages to screening for suppressors at 26oC and doing secondary screens to identify the subset of suppressors that had an associated temperature sensitive lethal phenotype.Thus, we did a second suppressor mutant screen to identify the TSC (Ts Suppressors of Ca Sensitivity) collection (5, 7, 8).Through the characterization of the TSC and SCS genes, we have identified and characterized many of the genes encoding proteins required for very long chain fatty acid (VLCFA) and long chain base (LCB) synthesis.
The long-term goal is to identify and characterize the proteins required for fatty acid elongation, to determine how the proteins are organized, to establish how synthesis of the VLCFAs is regulated, and to determine the essential functions of the VLCFAs.The immediate goal is to determine the specific functions of Elo2p, Elo3p, and Tsc13p, to identify the other elongating proteins, and to determine how the elongating proteins are organized.The elongase mutants provide the basis for biochemical and genetic studies that will identify other proteins required for VLCFA synthesis.The characterization of the proteins required for VLCFA synthesis is expected to advance the understanding of this important pathway, not only in yeast, but also in all eukaryotic cells.We have recently determined that TSC13 encodes the enoyl reductase (9), and that the YBR159 gene encodes the major 3-ketoreductase of the elongase system (10, 11).We are collaborating with Johnathan Napier in these studies.
Sphingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana. Chao DY, Gable K, Chen M, Baxter I, Dietrich CR, Cahoon EB, Guerinot ML, Lahner B, Lü S, Markham JE, Morrissey J, Han G, Gupta SD, Harmon JM, Jaworski JG, Dunn TM, Salt DE. Plant Cell. 2011 Mar;23(3):1061-81.
A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity.Gable K, Gupta SD, Han G, Niranjanakumari S, Harmon JM, Dunn TM. J Biol Chem. 2010 Jul 23;285(30):22846-52.
Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1. Eichler FS, Hornemann T, McCampbell A, Kuljis D, Penno A, Vardeh D, Tamrazian E, Garofalo K, Lee HJ, Kini L, Selig M, Frosch M, Gable K, von Eckardstein A, Woolf CJ, Guan G, Harmon JM, Dunn TM, Brown RH Jr. J Neurosci. 2009 Nov 18;29(46):14646-51.
Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. Han G, Gupta SD, Gable K, Niranjanakumari S, Moitra P, Eichler F, Brown RH Jr, Harmon JM, Dunn TM. Proc Natl Acad Sci U S A. 2009 May 19;106(20):8186-91.
Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals. Gupta SD, Gable K, Han G, Borovitskaya A, Selby L, Dunn TM, Harmon JM. J Lipid Res. 2009 Aug;50(8):1630-40.
Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-18:1 long chain base phosphate. Tsegaye Y, Richardson CG, Bravo JE, Mulcahy BJ, Lynch DV, Markham JE, Jaworski JG, Chen M, Cahoon EB, Dunn TM. J Biol Chem. 2007 Sep 21;282(38):28195-206.
In our laboratory we study redox-active metalloenzymes and their functions in microbial metabolism in anaerobic bacteria and archaea. Archaea are genetically distinct from eukaryotes and bacteria, and represent a unique domain of living organisms. Anaerobic bacteria and archaea impact human life in many ways; they play critical roles in global nitrogen and carbon cycles, they are useful to detoxify environmental and municipal wastes, and as part of the human gut microbiome they aid in digestion and the development of the immune system. Anaerobic microorganisms also can be pathogenic, and various species of Clostridia cause diseases such as tetanus, gangrene, acute ulcerative gingivitis, botulism and antibiotic-associated inflammatory diarrhea (C. difficile). Thus, research to reveal how specialized metalloenzymes function in anaerobic metabolism is useful to exploit the metabolic potential of these organisms for industrial, agricultural, and biomedical purposes.
Anaerobic methane producers, the methanogens, are the largest group of archaea, and we have been investigating the mechanism by which methanogens carry out large scale synthesis/cleavage of two-carbon acetyl units from one-carbon precursors/products. The reaction is catalyzed by a large 2,000 kDa multienzyme complex known as the ACDS complex (acetyl-CoA decarbonylase/synthase) that can make up as much as 25% of the soluble protein in the cell. Both the carbon-carbon and the carbon-sulfur bond of acetyl-CoA are formed/broken by the ACDS complex in a highly unusual biochemical mechanism involving metal-based carbonyl group insertion, and/or methyl group migration. Different catalytic roles of the five ACDS subunits include: CO dehydrogenase (αε), acetyl-CoA synthase (β) and B12 corrinoid methyltransferase (γδ) as shown below.
Scheme 1. ACDS complex partial reactions in the overall synthesis and cleavage of acetyl CoA. (H4SPt stands for tetrahydrosarcinapterin, a methanogen folate analog.)
We use molecular biological and biochemical techniques to explore the various reactions in this important multi-step process.
Recently, we carried out a side-by-side comparison of the enzymatic properties of the isolated archaeal subunit with the bacterial acetyl-CoA synthase, a subunit that is part of a smaller, two-subunit protein known as CO dehydrogenase/acetyl-CoA synthase (CODH/ACS). Our results revealed unexpected protein conformational control over the organometallic chemistry that takes place at the A cluster, a Ni- and Fe- containing active site metal center. Comparison of different ACS constructs showed acetyl C-C bond fragmentation was promoted by interdomain interactions involving the bacterial ACS N-terminal domain (a region of the protein not found in archaea). Our analyses of the role of a nearby phenylalanine residue showed remarkable effects on catalysis. We see that Nature provided the reactive Ni center with a repositionable aromatic shield, whose action is linked to the protein conformational state so as to selectively avoid CO substrate inhibition and to capitalize on the most productive sequence of steps in the reaction, maximizing the efficiency of acetyl-CoA synthesis. The enzyme thereby acquired the means to handle special requirements of an atypical organometallic mechanism, not found in organisms outside of anaerobic bacteria and archaea. A scheme that illustrates the individual steps and the connection with open/closed conformational states is shown below.
Scheme 2. Different modes of CO binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of Ni.
Our overall goal in studies on the mechanisms and functions of metalloenzymes is to provide a fundamental understanding of the unusual metabolic and physiological adaptations of important anaerobic microorganisms.
Gencic, S., Kelly, K., Ghebreamlak, S., Duin, E. C., and Grahame, D. A. (2013) Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel. Biochemistry 52, 1705-1716.
Grahame, D. A. (2011) Methods for analysis of acetyl-CoA synthase: Applications to bacterial and archaeal systems. Methods Enzymol. 494, 189-217.
Gencic S., Duin E.C., Grahame, D. A. (2010) Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila: Acetyl C-C bond fragmentation at the A cluster promoted by protein conformational changes." J. Biol. Chem. 285, 15450-15463.
Gencic, S. and Grahame, D. A. (2008) Two separate one-electron steps in the reductive activation of the A cluster in subunit beta of the ACDS complex in Methanosarcina thermophila. Biochemistry 47, 5544-5555.
Grahame, D. A., Gencic, S., and DeMoll (2005) A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl CoA in Methanosarcina thermophila. Arch. Microbiol. 184, 32-40.
We are working on understanding the mechanism of pre-mRNA splicing. Pre-mRNA splicing takes place in a large particle called the spliceosome, which consists of the pre-mRNA, five small nuclear RNAs (snRNAs), and a large number proteins. Splicing occurs via a two-step pathway. In the first step, the pre-mRNA the lariat intermediate and exon 1 are generated from the pre-mRNA. In the second step, the two intermediates are converted into the mRNA and released lariat intron. We have focused on the second step of splicing during which a transesterification reaction produces the final mRNA from the splicing intermediates. The second-step spliceosome is composed of the U2, U5, and U6 snRNPs together with many proteins; a number of proteins enter the spliceosome at specific stages following the first step, causing structural changes in the spliceosome that culminate in the second splicing reaction.
Our work has been centered on the Prp18 protein, which joins the spliceosome just prior to the transesterification reaction that generates the final spliced products. We showed that Prp18 acts in concert with the U5 snRNP during the second step of splicing and that an evolutionarily conserved region of Prp18 stabilizes the interaction of the splicing intermediates with the U5 snRNA. Analysis of the splicing of pre-mRNA substrates with mutations near the ends of the exons led us to propose a revised model of the interactions of the splicing intermediates with the U5 snRNA at the time of the second transesterification reaction (see Figure). In this model the exons are aligned to form a continuous helix that facilitates the second transesterification reaction. The model suggests that the interaction of exon1 with loop 1 of U5 changes from the first step to the second.
We are also studying the cyclophilin USA-CyP (PPi H). Cyclophilins have been studied intensively because of their medical importance as the targets of the immunosuppressive drug cyclosporin A, but the normal cellular functions of cyclophilins have been difficult to establish. USA-CyP was initially identified as a component of the U4/U6 snRNP through its strong association with the hPrp3 and hPrp4 proteins. We showed that USA-CyP forms stable complexes with two splicing factors, hPrp4 and hPrp18, and apparently enters the spliceosome separately within each of these complexes, as shown in the figure. USA-CyP functions in both steps of splicing, although it is not essential for splicing.
L. B. Crotti, D. Bacíková, and D. S. Horowitz, "Prp18 Stabilizes the Interaction of Both Exons with the U5 snRNA during the Second Step of pre-mRNA Splicing," Genes & Dev., 21, 1204-1216 (2007)
D. Bacíková and D. S. Horowitz, "Genetic and Functional Interaction of Evolutionarily Conserved Regions of the Prp18 Protein and the U5 snRNA," Mol. Cell. Biol., 25, 2107-2116 (2005)
D. Bacíková and D. S. Horowitz, "Mutational Analysis Identifies Two Separable Roles of the Saccharomyces cerevisiae Splicing Factor Prp18", RNA, 8, 1280-1293 (2002)
D. S. Horowitz, E. J. Lee, S. Mabon, and T. Misteli, "The Cyclophilin USA-CyP Functions in pre-mRNA Splicing," EMBO J, 21, 470-480 (2002) [highlighted in Science, 295, 931 (2002)]
M.S., Dept of Biology, Moscow State University, Moscow, Russia Ph.D., Shemyakin & Ovchinnikov Inst. of Bioorganic Chemistry, Moscow, Russia
Aneuploidy - the wrong number of chromosomes in an individual - is the leading cause of birth defects in humans. It results from errors in the segregation of homologous chromosomes (homologs) during gametogenesis. The proper segregation is ensured by meiotic recombination. It begins with the introduction of DNA double stranded breaks followed by their repair using the intact DNA of a homologous chromosome as a template. This leads to a temporal association of the homologs stabilized by crossing-overs. Such an arrangement into pairs ensures the orderly segregation of homologous chromosomes to the opposite poles of dividing nuclei so that each gamete receives one (and only one) homolog of each pair. The homologs that fail to pair segregate randomly, and have a 50% chance to go into the same daughter cell.
An estimated 10 to 30% of fertilized human eggs have the wrong number of chromosomes resulting in at least 5% of conceptions being aneuploid. Most of them abort before term making aneuploidy the leading known cause of pregnancy loss (~35% of miscarriages and ~4% of stillbirths). The number of aneuploid babies approaches 0.3% in newborns and those that survive face devastating consequences including developmental disabilities and mental retardation. Our long-term goal is to elucidate the mechanisms behind faulty meiotic recombination resulting in aneuploidy in mammals.
Both reduced recombination and abnormal location of recombination events are well-documented factors leading to aneuploidy. Therefore our research focuses both on the mechanisms that ensure optimal levels of homologous recombination as well as on the mechanisms that control the distribution of recombination events. We employ a wide range of approaches ranging from the biochemical characterization of purified proteins and the generation of genetically modified mice to the genome-wide characterization of the distribution of recombination events and the analysis of spatial organization of meiotic chromosomes.
Brick K, Smagulova F, Khil P, Camerini-Otero RD, Petukhova GV. Genetic recombination is directed away from functional genomic elements in mice. (2012) Nature, 485(7400): 642-645.
Khil, PP, Smagulova, F, Brick, KM, Camerini-Otero, RD., Petukhova, GV. Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA. (2012) Genome Res., 22(5): 957-65.
Smagulova F, Gregoretti IV, Brick K, Khil P, Camerini-Otero RD, Petukhova GV. Genome-wide analysis reveals novel molecular features of mouse recombination hotspots. (2011) Nature, 472(7343): 375-378.
Petukhova G., Pezza RJ, Vanevski F, Ploquin M, Masson JY and Camerini-Otero RD. The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination (2005) Nat Struct Mol Biol. 12(5), 449-53.
Petukhova G., Romanienko P, and Camerini-Otero RD. The Hop2 Protein has a Direct Role in Promoting Inter-Homolog Interactions during Mouse Meiosis. (2003) Dev. Cell 5(6), 927-936.
Petukhova G., Sung P, Klein H. Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1. (2000) Genes Dev. 14(17), 2206-15.
Petukhova G., S.A. Stratton, and P. Sung. Catalysis of Homologous DNA Pairing by Yeast Rad51 and Rad54 Proteins. (1998) Nature, 393:91-94.
Department of Primary Appointment: Biochemistry Position: Title: Associate Professor Biophysical chemistry of membranes;lipid-protein interactions; transfer of amphiphiles across and between membranes;peptide hydropathy
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Greenhut, Susan F. and Roseman, Mark A., "Cytochrome b5 Induced Flip- Flop of Phospholipids in Sonicated Vesicles", Biochemistry 24, 1252-1260 (1985).
Greenhut, S. F., Bourgeois, V. R., and Roseman, M. A., "Distribution of Cytochrome b5 Between Small and Large Unilamellar Vesicles", J. Biol. Chem. 261, 3670-3675 (1986)
Roseman, M. A., "Hydrophilicity of Polar Amino Acid Side Chains is Markedly Reduced by Flanking Peptide Bonds", J. Mol. Biol. 200, 513-522 (1988)
Grant, Jr., E., Beeler, T. J., Taylor, K. M. P., Gable, K., and Roseman, M. A., "Mechanism of Magainin 2a Induced Permeabilization of Phospholipid Vesicles," Biochemistry 31, 9912-9918 (1992).
Taylor, K. M. P., and Roseman, M. A., "The Effect of Cholesterol, Fatty Acyl Chain Composition, and Bilayer Curvature on the Interaction of Cytochrome b5 with Liposomes of Phosphatidylcholines," Biochemistry 34, 3841-50 (1995).
Ph.D in Life Sciences: Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India (affiliated to Jawaharlal Nehru University, New Delhi, India).
Post-doctoral research: Department of Molecular and Cellular Biology and Howard Hughes Medical Institute (HHMI), University of Arizona, Tucson, Arizona.
Role of Sm-like proteins in mRNA decay.
The long term goal of our lab is to understand the mechanism of mRNA decay in eukaryotes using the budding yeast S. cerevisiae as the model system. mRNA decay is a critical determinant of gene expression and deregulation of mRNA decay is known to be the cause of several human diseases including cancer.
The mRNA decay pathways and decay factors are well conserved in all eukaryotes from yeast to humans. Two major pathways of mRNA decay exist in eukaryotes. Both pathways are initiated by poly(A) shortening of the mRNA. In the 5’ to 3’ pathway, this is followed by decapping which then permits the 5’ to 3’ exonucleolytic degradation of the message body. In the 3’ to 5’ decay pathway, deadenylated mRNAs are degraded in a 3’ to 5’ exonucleolytic manner.
In the 5’ to 3’ pathway, decapping is a crucial precisely controlled step affected by numerous factors. Oligoadenylated mRNAs but not polyadenylated mRNAs are selectively targeted for decapping in the 5’ to 3’ decay pathway resulting in the deadenylation dependence of decapping in this pathway. While translation initiation factors are antagonistic to the decapping enzyme, several other factors enhance the decapping enzyme function in vivo.
The Lsm1p-7p-Pat1p complex (made up of seven Sm-like proteins, Lsm1 through Lsm7 which are characterized by the presence of the Sm-domain and the Pat1 subunit) is a key activator of decapping needed for normal rates of decapping in vivo. It is conserved in all eukaryotes and interacts with several decay factors and with the mRNA in vivo. Interestingly, this complex selectively associates with oligoadenylated mRNPs targeted for decapping in vivo.
We purified the native Lsm1-7-Pat1 complex from yeast and showed that it intrinsically has a higher affinity for oligoadenylated RNA over polyadenylated RNAs in vitro. Importantly loss of such ability to recognize the oligo(A) tail due to mutations in the Sm domain of Lsm1 impairs mRNA decay function of this complex in vivo. By studying multiple lsm1 mutants we also showed that decapping activation by the Lsm1-7-Pat1 complex in vivo requires both the binding of that complex to the mRNA and facilitation of one or more (unknown) post-binding events.
As mentioned above the residues in the Sm-domain of Lsm1 are crucial for the in vivo functions and unique in vitro RNA binding properties of the Lsm1-7-Pat1 complex. However, unlike many other Sm-like proteins, yeast Lsm1 has a long C-terminal domain (CTD) following its Sm-domain and this feature is conserved in human Lsm1 also. Interestingly our studies showed that the CTD of Lsm1 is also necessary (in addition to the Sm-domain of Lsm1) for the in vivo functions and the RNA binding activity of the Lsm1-7-Pat1 complex. Further, the CTD of Lsm1 could even act in trans to support the function of the Lsm1-7-Pat1 complex in vivo suggesting that it folds as a separate domain in the Lsm1 subunit. Thus Lsm1 is a unique Sm-like protein whose functions are determined not just by its Sm-domain but also residues outside the Sm-domain.
Additional studies showed that the Pat1 subunit is also critical for the in vivo functions and unique in vitro RNA binding properties of the Lsm1-7-Pat1 complex like the Lsm1 subunit and that the RNA binding surface(s) of the Lsm1-7-Pat1 complex are probably composite including residues from both the Lsm1-7 assembly and the Pat1 subunit.
The Lsm1-7-Pat1 complex has also been implicated in viral RNA translation. Our recent studies using lsm1 mutants and the yeast system for replication of Brome Mosaic Virus (BMV) have revealed that the Lsm1-7-Pat1 complex binds to different regions of the BMV genomic RNAs and promote their translation and recruitment out of translation to replication via different mechanisms.
NIH RO1 grant (GM072718).
USUHS exploratory grant (R071JX).
Chowdhury, A, Swathi Kalurupalle, S, Tharun, S. 2016. Mutagenic Analysis of the C-Terminal Extension of Lsm1. PLoS ONE, 11: e0158876.
Jungfleisch, J, Chowdhury, A, Alves-Rodrigues, I, Tharun, S.*, Díez, J. 2015. The Lsm1-7- Pat1 complex promotes viral RNA translation and replication by differential mechanisms. RNA, 21:1469–1479 (*Corresponding author).
Chowdhury, A., Kalurupalle, S., and Tharun, S. 2014. Pat1 contributes to the RNA binding activity of the Lsm1-7- Pat1 complex. RNA, 20:1465–1475.
Chowdhury, A., Raju, KK., Kalurupalle, S., and Tharun, S. 2012. Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1–7–Pat1 complex. RNA, 18:936–944.
Chowdhury, A., and Tharun, S. 2009. Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7- Pat1 complex. RNA, 15:1837–1848.
Tharun, S. 2009. Lsm1-7- Pat1 complex: A link between 3’ and 5’-ends in mRNA decay? RNA Biology, 6(3):228-232.
Tharun, S. 2009. Roles of eukaryotic Lsm proteins in the regulation of mRNA function. International Review of Cell & Molecular Biology, 272:149-89.
Tharun, S. 2008. Purification and analysis of the decapping activator Lsm1p-7p- Pat1p complex from yeast. Methods in Enzymology, 448:41-55.
Chowdhury, A., and Tharun, S. 2008. lsm1 mutations impairing the ability of the Lsm1p-7p- Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo. RNA, 14:2149-2158.
Chowdhury, A., Mukhopadhyay, J., Tharun, S. 2007. The decapping activator Lsm1p-7p- Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs. RNA, 13:998-1016.
Tharun, S.*., Muhlrad D., Chowdhury A, Parker R. 2005. Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection. Genetics, 170:33-46 (*Corresponding author).
Tharun, S.*, and Parker R. 2001. Targeting an mRNA for decapping: Displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Molecular Cell, 8:1075-1083 (*Corresponding author).
Tharun, S., He, W., Meyes, A., Lennertz, P., Beggs, J., and Parker, R. 2000. Yeast Sm-like proteins function in mRNA decapping and decay. Nature, 404:515-518.
Tharun, S., and Parker, R. 1999. Analysis of mutations in the yeast mRNA decapping enzyme. Genetics, 151:1273-1285.
Research in my lab is to study potential drug target proteins encoded in the genome of Mycobacterium tuberculosis (MTB), the causative agent of the disease tuberculosis. We use X-ray crystallographic techniques and in vitro biochemical assays to analyze the functions, reaction mechanism, and protein-protein interactions of these proteins. Current projects include studying the PhoP-PhoR two-component signaling system, and an iron-regulated ABC-type transporter IrtAB.
The PhoP-PhoR two-component system is essential for virulence and intracellular growth of MTB. Global profiling of gene expression indicates that at least 44 genes are up-regulated and 70 genes are down-regulated by PhoP-PhoR. A mutant MTB lacking this two-component system has defects in cell envelope, and it cannot grow in human and mouse macrophages or in mice. PhoR is a sensor histidine kinase that transmits environmental signals through cell membrane by autophosphorylation on its cytosolic domain. The phosphate group is then transferred to PhoP, a response regulator, to activate its regulation of gene transcription. We are studying the structures of the PhoP and PhoR proteins, the interactions between them, the mechanism of PhoR autophosphorylation and phosphorylation of PhoP, and the DNA recognition mechanism of PhoP.
PhoP has two distinct domains, an N-terminal regulatory domain (also called receiver domain) that contains the phosphorylation site aspartate and a C-terminal DNA-binding domain (also called effector domain). The crystal structure of PhoP indicates that it can dimerize through the receiver domain to form a symmetric dimer. The effector domains of the dimer are tethered to the N-terminal domains through a flexible linker, but do not have any interactions between themselves or with the receiver domains in the dimer. While the sequence recognition helix is exposed without phosphorylation activation and is able to bind DNA, phosphorylation is likely to stabilize the receiver domain dimer and thus increase binding affinity to tandem repeat DNA sequences by bringing two effector domains in close proximity.
PhoR has a modular domain structure: an extracytosolic sensor domain (PhoRE), a transmembrane domain (PhoRTM), and a cytosolic domain (PhoRC). The cytosolic domain can be subdivided into a HAMP domain (PhoRH), a dimerization domain (PhoRDD), and an ATPase domain (PhoRA). A truncated domain (PhoRK) containing both PhoRA and PhoRDD is expected to have the kinase activity. We are working on these truncated domains, as well as the full-length PhoR protein.
The genes irtA and irtB are regulated by iron and are in a single operon. IrtA and IrtB are predicted to form a heterodimeric ABC-type transporter essential for import of iron-siderophore complex under low iron conditions. There is an atypical extension of ~290 residues at the N-terminus of IrtA, which is predicted to bind siderophore based on sequence analysis. This extension is termed SID for siderophore interacting domain. SID expressed and purifed from E. coli has a bright yellow color from its bound cofactor FAD. Because SID is located in the cytosol, it is likely to function as a ferrireductase to release iron from its siderophore complex.
X. He and S. Wang. DNA Consensus Sequence Motif for Binding Response Regulator PhoP, a Virulence Regulator of Mycobacterium tuberculosis. Biochemistry (2014), 53, 8008-20.
N. Sambuughin, X. Liu, S. Bijarnia, T. Wallace, I.C. Verma, S. Hamilton, S. Muldoon, L.J. Tallon and S. Wang. Exome sequencing reveals SCO2 mutations in a family presented with fatal infantile hyperthermia. J. Hum. Genet. (2013) 58, 226-8.
S. Wang. Bacterial Two-Component Systems: Structures and Signaling Mechanisms, in Protein Phosphorylation in Human Health, p439-466, (2012), C. Huang (Ed.), ISBN: 978-953-51-0737-8, InTech, DOI: 10.5772/48277.
S. Menon and S. Wang. Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer through the receiver domain. Biochemistry (2011), 50, 5948-5957.
M. B. Ryndak, S. Wang, I. Smith, and G. M. Rodriguez. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding Domain. J. Bacteriol. (2010) 192, 861-869.
M. Ryndak, S. Wang, and I. Smith. PhoP, a key player in Mycobacterium tuberculosis virulence. Trends in Microbiology (2008) 16, 861-869.
S. Wang, J. Engohang-Ndong, and I. Smith. Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis. Biochemistry (2007) 46, 14751-14761.
S. Wang and D. Eisenberg. Crystal Structure of the Pantothenate Synthetase from Mycobacterium tuberculosis, Snapshots of the Enzyme in Action. Biochemistry (2006) 45, 1554-1561.
M. Strong, M. Sawaya, S. Wang, M. Phillips, D. Cascio and D. Eisenberg. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. (2006) 103, 8060-8065.
S. Wang and D. Eisenberg. Crystal Structures of a Pantothenate Synthetase from M. tuberculosis and its complexes with substrates and a reaction intermediate. Protein Science (2003) 12, 1097-1108.
S. Wang, C. Mura, M. Sawaya, D. Cascio and D. Eisenberg. Crystal Structure of a Nudix Protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit ? sheets. Acta Cryst. (2002) D58, 571-578.
S. Wang, L. Tabernero, M. Zhang, E. Harms, R. L. Van Etten and C. V. Stauffacher. Crystal Structures of a Low Molecular Weight Protein Tyrosine Phosphatase from Saccharomyces cerevisiae and its Complex with the Substrate p-Nitrophenyl Phosphate. Biochemistry (2000) 39, 1903-1914.
S. Wang, C. V. Stauffacher and R. L. Van Etten. Structural and Mechanistic Basis for the Activation of a Low Molecular Weight Protein Tyrosine Phosphatase by Adenine. Biochemistry (2000) 39, 1234-1242.
B.S. & M.S. Department of Biology, Peking University, Beijing, P. R. China
Ph.D. Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey. Advisor: Dr. Kiran Chada.
Post-doctoral research: Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey. Advisor: Dr. Ron Morris.
Cytoplasmic dynein is a microtubule motor protein that transports a variety of cargoes including vesicles/organelles, proteins and mRNAs. Its proper function is crucial for human health as mutations in dynein or its regulators, such as dynactin and Lis1, are causally linked to brain developmental disorders and neurodegenerative diseases. We are interested in how dynein attaches to its cargoes and how dynein activity is coordinated with cargo binding.
Weuse the filamentous fungus Aspergillus nidulans as a genetic model organism for our studies. In A. nidulans, dynein accumulates at the microtubule plus end near the hyphal tip in a kinesin-1- and dynactin-dependent manner, which places the dynein motor in close proximity with the early endosome cargo. A few years ago we found that the dynein-early endosome interaction in A. nidulans specifically requires the p25 subunit of dynactin. Based on the phenotype of the p25 null mutant, we performed a genetic screen to uncover the unknown molecular machinery linking dynein-dynactin to early endosomes.
Our genetic approach has allowed us to identify two key proteins, HookA (Hook in A. nidulans) and FhipA (FHIP in A. nidulans), which link dynein-dynactin to early endosomes. FhipA is required for the C-terminus of HookA to bind early endosome. The N-terminal part of HookA is important for the HookA-dynein-dynactin interaction, which intriguingly requires not only dynactin p25 but also the dynein complex. We are currently dissecting the role of p25 in HookA binding and dynactin regulation. Recently, we have also discovered a vezatin-like protein VezA as a novel factor crucial for dynein-mediated early endosome transport. Interestingly, VezA is not a cargo adapter like HookA or FhipA, but instead, it localizes at the hyphal tip and regulates the interaction between the HookA-bound eary endosomes and dynein-dynactin. The mechanism of VezA action remains a mystery and we will elucidate it using a combination of biochemical and genetic approaches.
Current lab members
Jun Zhang Research Assistant Professor B.S. Shanghai Medical University Ph.D. Beijing Medical University email@example.com
Rongde Qiu Research Associate B.S. Fudan University M.S. Institute of Biochemistry & Cell Biology, SIBS, CAS firstname.lastname@example.org
Xuanli (Lia) Yao Research Assistant Professor B.S. Nanjing University Ph.D. Ohio State University
Selected Recent Publications
1.Yao, X., Arst, H.N. Jr, Wang, X. and Xiang, X. (2015). Discovery of a vezatin-like protein for dynein-mediated early endosome transport. Mol. Biol. Cell. 26, 3816-3827. (highlighted by MBoC Highlights)
2.Wang, B., Li, K., Jin, M., Qiu, R., Liu, B., Oakley, B.R. and Xiang, X. (2015). The Aspergillus nidulansbimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors. Fungal Genet. Biol. 82:51-55.
3.Pantazopoulou, A., Pinar, M., Xiang, X., Peñalva, M.A. (2014) Maturation of late Golgi cisternae into RabERAB11 exocytic post-Golgi carriers visualized in vivo. Mol. Biol. Cell. 25, 2428-2443.
4.Yao, X., Wang, X., Xiang, X. (2014) FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus. Mol. Biol. Cell. 25, 2181-2189. (highlighted by MBoC Highlights)
6.Zhang, J., Twelvetrees, A.E., Lazarus, J.E., Blasier, K.R., Yao, X., Inamdar, N.A., Holzbaur, E.L.F.*, Pfister, K.K.*, Xiang, X.* (2013) Establishing a novel knock-in mouse line for studying neuronal cytoplasmic dynein under normal and pathologic conditions. Cytoskeleton. 70, 215-227.
7.Qiu, R., Zhang, J., Xiang, X. (2013) Identification of a novel site in the tail of dynein heavy chain important for dynein function in vivo. J. Biol. Chem. 288, 2271-2280.
8.Yao, X., Zhang, J., Zhou, H., Wang, E., Xiang, X. (2012) In vivo roles of the basic domain of dynactin p150 in microtubule plus-end tracking and dynein function. Traffic 13, 375-387.
9.Zhang, J.*, Yao, X.*, Fischer, L. Abenza J. F., Peñalva. M. A., Xiang, X. (2011) The p25 subunit of the dynactin complex is required for dynein-early endosome interaction. J. Cell Biol. 193, 1245-1255.
10.Zhang, J., Tan, K., Wu, X., Chen, G., Sun, J., Reck-Peterson, S., Hammer, J.A. III*, Xiang, X.* (2011) Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport. PLoS ONE, 6(12):e28575.
11.Zhai, B*., Zhou, H.*, Yang, L., Zhang, J., Jung, K., Giam, C.Z., Xiang, X., Lin X. (2010) Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect. J Antimicrob Chemother. 65, 931-938.
12.Zhang, J., Zhuang, L., Lee, Y. Abenza J. F., Peñalva. M. A., Xiang, X. (2010) The microtubule-plus-end localization of Aspergillus dynein is important for dynein-early endosome interaction but not for dynein ATPase activation. J. Cell Sci. 123, 3596-3604.
(* co-first or co-corresponding authors)
Invited Reviews/Book Chapters
1.Xiang, X, Qiu, R., Yao, X., Arst, H.N. Jr, Peñalva, M.A. and Zhang, J. (2015). Cytoplasmic dynein and early endosome transport (review). Cellular and molecular life sciences 72, 3267-3280.
2.Xiang, X. (2012) Nuclear Positioning: dynein needed for microtubule shrinkage-coupled movement (dispatch). Curr. Biol. 22, R496-499.
3.Xiang, X. (2011) Insights into Cytoplasmic Dynein Function and Regulation from Fungal Genetics (book chapter). In Dyneins: Structure, Biology and Disease (Edited by S. M. King), Elsevier. p455-481
4.Xiang, X. and Oakley, B. R. (2010). The Cytoskeleton in Filamentous Fungi (book chapter). In: Cellular and Molecular Biology of Filamentous Fungi (Edited by K. Borkovich and D. Ebbole), ASM Press. p209-223.
Post-doctoral research. University of Michigan, Life Sciences Institute
(Janet L. Smith) and Department of Biophysics (Martha L. Ludwig)
PhD. University of Michigan, Department of Chemistry (Dimitri Coucouvanis)
Diploma. University of Athens, Department of Chemistry
The research in my lab aims to understand biologically important processes by revealing the complex relationships between the structure, function, and dynamics of proteins through a structural enzymology approach. We take advantage of a combination of x-ray crystallography, biochemical, kinetic, and thermodynamic techniques to describe the functional pathway of biologically relevant molecules. Specifically, my lab focuses on proteins in three processes essential to human health:
Homocysteine (Hcy) is an essential metabolite critical for methionine, S-adenosylmethionine (SAM) and cysteine biosynthesis. Elevated levels of Hcy (hyperhomocysteinemia) pose an increased risk for a number of diseases including cardiovascular diseases, making the regulation of cellular Hcy vital to human health. In mammals, four enzymes control Hcy homeostasis: methionine synthase (MS), cystathionine β-synthase (CBS), and betaine-homocysteine S-methyltransferase (BHMT and BHMT2). My lab studies the structure and function of all four of these enzymes.
Fig. 1 Homocysteine metabolic pathway.
In the one carbon (methionine) cycle Hcy is a product of the transmethylation reactions. Hcy either enters the transsulfuration pathway (via CBS) to eventually form glutathione, or is remethylated to methionine by MS or BHMT in the one carbon cycle. MS,couples Hcy metabolism to the folate cycle.
Methionine synthase (MS). Methionine synthase is a multi-domain vitamin B12-dependent enzyme whose function relies on the delivery of vitamin B12 through a complex B12 maturation/delivery pathway. In mammals, MS is the only Hcy clearing enzyme expressed in all tissue types, and the only such enzyme found in vascular tissue. MS polymorphisms are linked to numerous human diseases and cause megaloblastic anemia. However, the function and regulation of mammalian MS are poorly understood. Moreover, it is unclear how MS polymorphisms disrupt enzyme function. The goal of this work is to reveal the mechanism of human MS catalysis and regulation, providing insight into a variety of MS and Hcy-related human diseases. This work will characterize the relationship between MS function and B12 metabolism, and provide a framework for understanding how MS contributes to Hcy homeostasis. Our studies will reveal which B12 maturation/delivery proteins might make suitable targets for new therapeutics against the cardiovascular disease risk factor hyperhomocysteneimia.
Betaine-homocysteine S-methyltransferases (BHMT and BHMT2). BHMT and BHMT2 are zinc dependent mammalian enzymes that catalyze the same reaction as MS, but with remarkably different chemistry and protein scaffolds. In addition to maintaining Hcy levels, these proteins are postulated to contribute to lipid and phospholipid metabolism. Through a combination of structural enzymology, chemical biology, and in vivo approaches, we are working to uncover the structure, mechanism, and precise physiological roles of BHMT and BHMT2 in vivo.
Fig. 2Structure of CBS from Drosophila shown in a dimer pair.
Cysteine (Cys) biosynthesis via the transsulfuration pathway is an integral piece of the Hcy degradation pathway. The production of Cys is not only essential for Hcy catabolism, but also for the synthesis of glutathionine (GSH), an abundant small thiol molecule critical for the protection of cells from oxidative damage and maintaining redox homeostasis. The transsulfuration of Hcy to Cys is catalyzed by the successive action of two enzymes: cystathioninine b-synthase (CBS) and cystathionine g-lyase (CSE). Mutations in CBS are the single most common cause of hereditary hyperhomocysteinemia with > 100 mutations having been observed in patients. In addition to converting Hcy to Cys, these two enzymes are responsible for the biosynthesis of the newly discovered signaling molecule H2S, which is important for the cardiovascular and nervous systems. Recently, a third enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), was identified as an additional contributor to H2S production. H2S serves a number of roles such as acting as a neuromodulator, regulator of hormone release, vasorelaxative, preserver of mitochondrial function, endogenous modulator of inflammation, and increasing levels of GSH by activating cystine transport. Unlike other gasotransmitters (eg. CO and NO) the vascular effects, mechanism of action, synthesis, and regulation of H2S are not well studied. The aim of this project is to understand the fundamentals of H2S biosynthesis and regulation through the investigation of the enzymes of the transsulfuration pathway involved in its production.
Mitochondrial (mt) tRNA genes are hot spots for mutations that lead to adverse health effects with a wide range of pathology, from isolated organ-specific diseases such as myopathy or hearing loss, to multisystem disorders with encephalopathy, gastrointestinal dysmotility, and life-threatening cardiomyopathy. All tRNAs are regularly interspersed throughout the entire compact mitochondrial genome and require the combined action of two mitichondrial endonucleases, a 5' and 3' end one in order to be excised. Mitochondrial tRNA processing is important not only for the maturation and release of tRNAs but also of for the maturation and release of of rRNAs and mRNAs. Mutations in tRNA genes may impact multiple steps during a tRNA's life cycle, including 5' end maturation by mitochondrial ribonuclease P (mtRNaseP). RNase P is responsible for catalyzing tRNA 5' end maturation across all three domains of life. Our goal is to investigate this maturation process since we believe it provides a mechanistic link to mitochondrial dysfunction and aging.
Fig. 3 Structure of mt PNase P from Arabidopsis thaliana with precursor tRNA substrate modeled in.
Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P (mt RNase P or MRPP3) from human mitochondria and A. thaliana chloroplast and mitochondria shifted this paradigm, with these enzymes representing a new class of metallonucleases. MRPP3s are conserved among higher eukaryotes and evolved recently as yeast mitochondrial genomes encode RNase P with a catalytic RNA. We recently solved the structure of mt RNase P from A. thaliana revealing an N-terminal PPR domain and a C-terminal metallonuclease PIN-like domain. In higher eukaryotes two additional proteins were identified as important for in vivo MRPP3 activity (MRPP1 and MRPP2). We hypothesize that the three MRPP proteins are essential for mitochondrial viability and provide a link between RNA processing and mitochondrial diseases and aging. We are employing a highly collaborative in vivo and in vitro approach to study the structure, mechanism and role of MRPPs in biological processes.
Howard MJ. Lim W, Fierke CA, Koutmos M. Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5'-processing. (2012) Proc Natl Acad Sci., in press
Koutmos M, Gherasim C, Smith JL, Banerjee R. Structural basis of multifunctionality in a vitamin B12-processing enzyme. (2011) J Biol Chem. 286(34)
Koutmos M, Kabil O, Smith JL, Banerjee R. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine b-synthase. (2010) Proc Natl Acad Sci. 107(49)
Hsieh J, Koutmou KS, Rueda D, Koutmos M, Walter NG, Fierke CA. A divalent cation stabilizes the active conformation of the B. subtilis RNase P·pre-tRNA complex: a role for an inner-sphere metal ion in RNase P. (2010) J Mol Biol. 400(1)
Koutmos M, Datta S, Pattridge KA, Smith JL, Matthews RG. Insights into the reactivation of cobalamin-dependent methionine synthase. (2009) Proc Natl Acad Sci. 106(44)
Koutmos M, Pejchal R, Bomer TM, Matthews RG, Smith JL, Ludwig ML. Metal active site elasticity linked to activation of homocysteine in methionine synthases. (2008) Proc Natl Acad Sci. 105(9)