Abstracts Submitted: Division III - Graduate students (2 + years) - Biomedical Sciences
CHARACTERIZATIONS OF CONFORMATIONAL CHANGES AND SINGLE-STRANDED DNA BINDING PROPERTIES OF REPLICATION PROTEIN A AFTER HYPERPHOSPHORYLATION BY DNA-PK
Yiyong Liu and Yue Zou, Department of Biochemistry and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614
Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein necessary for DNA replication, repair, and recombination, as well as cellular DNA-damage response. RPA is a heterotrimer consisting of subunits of 70, 32, and 14 kDa. It has been well reported that the N-terminus of the middle subunit RPA32 becomes hyperphosphorylated by phosphatidyl inositol 3-kinase-like serine/threonie protein kinases (PIKKs) including DNA-dependent protein kinase (DNA-PK) after cellular DNA damage. The hyperphosphorylated RPA changes its interactions with proteins and DNA, suggesting changes of its activities and conformation upon hyperphosphorylation. In the present study, we prepared a type of hyperphosphorylated RPA (hyp-RPA) by in vitro phosphorylation of RPA with DNA-PK. The hyp-RPA has a same mobility shift on SDS-PAGE as form 5 of the RPA in UV-irradiated HeLa cells. MS/MS analysis showed that five sites in RPA32N, including Ser-4, Ser-23, Ser-29, Thr-21 and Ser-33, were phosphorylated in the hyp-RPA. Chemical modification combined with comparative mass spectrometric analysis revealed that no dramatic conformational changes occur in RPA upon hyperphosphorylation, but the chemical reactivities of the residues on one arm (amino acids 330-345) of the cleft of DNA-binding domain B (DBD-B) were diminished, indicating a direct interaction between hyperphosphorylated RPA32N and the cleft of DBD-B. This proposal is supported by RPA proteolysis assays. The binding of hyp-RPA to short ssDNA or partial DNA duplex containing 5’ or 3’-protruding short ssDNA was reduced when compared with the binding of nonphosphorylated RPA. The changes of conformation and DNA-binding activities of RPA upon hyperphosphorylation characterized here may have physiological significance in DNA metabolism.
Cerrone Foster and Scott Champney, Department of Biochemistry, East Tennessee State University, College of Medicine, Johnson City, TN
The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of the assembly intermediate produced in E.coli cells treated with neomycin or paromomycin. Escherichia coli cells were grown with neomycin or paromomycin and labeled with 3H-uridine. Cell lysates of drug treated cells were separated by sucrose gradient centrifugation. The size and content of ribosomal RNA from different regions was determined by hybridization studies. 30S subunit ribosomal proteins labeled with 35S-methionine were collected from 30S subunits and the 21S precursor particle. Two dimension gel electrophoresis was performed to identify the protein content of the precursor particle from cells treated with each antibiotic. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentiation coefficient of 21S. Hybidization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Fourteen 30S ribosomal proteins were found in the precursor after inhibition by each drug. The precursor particle was also shown to bind radio-labeled antibiotics during cell growth. The two aminoglycosides examined prevented 30S assembly and stimulated the formation of a 21S precursor containing 17S rRNA and 14 ribosomal proteins. The results indicate important features of a novel target for aminoglycosides in cells.
Brian D. Dill 1, Rick W. LaRue 1, Jane E. Raulston 1, 2 Department of Microbiology 1, Department of Pathology 2 Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614
Iron is vital to numerous, highly conserved metabolic cellular processes. Numerous bacterial virulence factors exist to supply pathogens with iron in mammalian hosts, where availability is tightly controlled to avoid the generation of free radicals via the Fenton reaction. Previous studies have shown Chlamydia trachomatis requires iron for growth and viability. The purpose of this study is to identify chlamydial proteins that increase in expression following iron chelation, with a focus on the multiple heat shock proteins. Chlamydial Hsp60 has been implicated in the more severe sequelae of chlamydial disease. Human endometrial carcinoma (HEC-1B) cells were infected with C. trachomatis serovar E. Cells were exposed to the eukaryotic protein synthesis inhibitor cycloheximide, and desferal (an iron chelator) was added to the medium during the logarithmic stage of chlamydial growth. Total cell lysates were resolved by SDS-PAGE or two-dimensional (2D) PAGE, and Western blots were probed using monospecific, polyclonal antibodies against Hsp60-1, -2, -3, and Hsp70. Results indicate that Hsp60-2 is strongly responsive to iron limitation, whereas Hsp60-3 and Hsp70 show little to no difference in expression levels; Hsp60-1 is slightly responsive to iron early in chlamydial infection. The response of Hsp60-2 to iron deprivation occurs within 30 minutes, indicating chlamydiae are capable of a rapid response to iron deprivation.
Marie-Rose A. Abdo, BS, MS, Mary-Katherine Anderson, PhD, RN, FNP, Jamie Branam Kridler, PhD, Elizabeth Lowe, MS, RD, LDN
Cardiovascular disease (CVD) is the most common cause of death in the United States. In 2002, it was estimated that nearly seventy million Americans had some form of CVD. At least 62 million Americans have CVD, and over two and a half million die each year from the disease. Older adults are at higher risk for chronic illnesses and are interested in learning more about their own health. With demographic changes and an increase in the number and proportion of older adults, there is now a greater demand for health promotion and patient education by rehabilitation professionals including cardiac rehabilitation. Despite the apparent benefit of cardiac rehabilitation, it has been shown that only 11% to 38% of males and females actually participate in rehabilitation programs, which represents about one-third of those eligible. The purpose of this study was to determine if cardiac nutrition education can affect dietary patterns of subjects participating in Cardiac Rehabilitation. Surveys, a pretest given prior to the first cardiac class and a second identical test, administered following cardiac rehabilitation classes, were conducted. Fifty-five randomly selected participants from the outpatient Cardiac Rehabilitation Program were initially given the posttest. Thirty-three of the 55 surveys were returned. Cardiac health classes offered by various health professionals included cardiac nutrition education, group nutrition classes, guest lectures on nutrition, and cooking demonstrations. The tests were compared to measure changes in diet and were analyzed for both surveys. Paired t-tests were used for analysis along with frequency, percentage and group statistics. The majority of the respondents demonstrated a statistically significant healthy increase in dietary changes after having participated in the classes. This confirms that cardiac rehabilitation programs play an important role in individuals with heart disease as a tertiary prevention. This has been proven in other studies to decrease the risk of having another cardiovascular event. Providing nutrition classes within the cardiac rehabilitation program was found to be an effective way to help the subjects understand what causes cardiovascular disease and follow the ways to prevent further complications in their disease; this is particularly important when CVD is, and has been for the last few decades, the leading cause of death in the United States.
Giles, D.K. 1, R.W. LaRue 1, P.B. Wyrick 1, and J.E. Raulston 1,2 , Departments of Microbiology 1 and Pathology 2, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614
Several studies have identified an association between chlamydial heat shock protein 60 (chsp60) and the deleterious sequelae of genital chlamydial infection, including tubal infertility, pelvic inflammatory disease, and salpingitis. Whole genome sequencing of C. trachomatis revealed two hsp60 homolog proteins in addition to groEL-1. Since chsp60 has been implicated as a critical antigen responsible for stimulating immune-mediated inflammation and disease, the expression, distribution, and function of the three copies of hsp60 are of great interest. Transcriptional analysis has shown that the expression of these genes is independent of one another and differential expression is observed in active vs. persistent infection. Our objective was to determine, using specific peptide antisera for each copy of chsp60, the bacterial and host cellular location(s) and distribution of chsp60-1,2,3 during active, persistent, and azithromycin-exposed chlamydial infection. Under normal conditions, chsp60-1,2,3 all exhibited similar distribution within both elementary bodies (EB) and reticulate bodies (RB), including membrane association. During penicillin and azithromycin treatment, chsp60-2 and -3, but not chsp60-1, were detected in both chlamydial membrane blebs and within everted inclusion membrane vesicles. Likewise, antibiotic exposure resulted in the visualization of chsp60-2 and -3, but not chsp60-1, at higher levels free in the inclusion, at the cell surface, and released extracellularly. Our results found that chsp60-2 is qualitatively elevated at the protein level during persistent infection. Thus, localization of the three copies of chsp60 during stressful and cidal conditions may provide more clues about chlamydial pathogenesis as it relates to immune recognition and response.
INTERLEUKIN-1 β DECREASES VASCULAR ENDOTHELIAL GROWTH FACTOR-D EXPRESSION AND AFFECTS ANGIOGENESIS IN CARDIAC MICROVASCULAR ENDOTHELIAL CELLS
Deidra J.H. Mountain, Mahipal Singh, Ph.D., and Krishna Singh, Ph.D., Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, James H. Quillen Veterans Affairs Medical Center, Johnson City, TN 37614
Angiogenesis is essential in the repair of the heart following myocardial infarction (MI). Angiogenesis involves a cascade of events including extracellular matrix (ECM) degradation, endothelial cell (EC) migration and proliferation, and capillary tube formation. Interleukin-1 β (IL-1 β ), increased in the heart post MI, plays a role in ECM remodeling and is considered pro-angiogenic in cells of non-cardiac origin via the involvement of vascular endothelial growth factors (VEGFs). VEGFs promote EC proliferation and migration. Polymerization of actin plays a critical role in tube formation and vascular stability. In the present study we investigated IL-1 β -mediated expression of various angiogenic genes, studied the signaling pathways involved in the regulation of VEGF-D, and examined the angiogenic role of IL-1 β in cardiac microvascular endothelial cells (CMECs). Methods: Primary cultures of CMECs, isolated from adult rat hearts, were exposed to IL-1 β (4 ng/ml) for 24h. Total RNA was reverse transcribed and resulting cDNAs were hybridized with a gene array membrane containing 96 angiogenesis-related genes. Western blot analyses were used to examine the expression of VEGF-D and activation of MAPKs (mitogen activated protein kinases; ERK1/2 and JNKs). Wound healing assays, performed by scraping confluent cultures with a pipette tip, were used to measure cell migration. Cell proliferation was measured by seeding 2.0x 10 4 cells per dish and manually counting the cells following 24h exposure to IL-1 β . Actin-polymerization was studied using Phalloidin-FITC stain, specific for F-actin. Results: Gene array analysis indicated that IL-1 β decreases the expression of VEGF-D, not VEGF-A. This IL-1 β -mediated decrease in VEGF-D expression was confirmed by western blot analysis, where IL-1 β decreased VEGF-D protein levels by 48% (p<0.05 vs. control). IL-1 β activated ERK1/2 and JNKs. UO126 (10 µ M; inhibitor of ERK1/2 pathway) and SP600125 (10 µ M; inhibitor of JNKs) partially but significantly inhibited the IL-1 β -mediated decrease in VEGF-D expression. SN50 (10 μ M; inhibitor of NF κ B) and chelerythrine (2 μ M, inhibitor of PKC) exhibited synergistic effects with IL-1 β by further decreasing VEGF-D protein levels by 42% and 47%, respectively (p<0.05 vs. IL-1 β ). Inhibitors alone had no effect. IL-1 β promoted cell migration, decreasing the wound distance by 82.5 µ m after 24h (p<0.05 vs. time 0,~210 µ m). However, IL-1 β did not allow cell-to-cell contact in the wound healing assays. Interestingly, treatment of cells with VEGF-D promoted cell migration, cell-to-cell contact, and complete healing of the wound within 24h. IL-1 β failed to significantly affect cell proliferation. Actin polymerization, indicative of tube formation, was significantly reduced by IL-1 β (p<0.01 vs. control). Conclusion: IL-1 β may play an anti-angiogenic role in the heart post MI by inhibiting expression of VEGF-D and tube formation. With regard to IL-1 β , strategies for optimizing regrowth of functional blood vessels in the ischemic myocardium would be of great clinical importance.
Timothy R. Southern, Melissa E. Lester, and J. Russell Hayman, Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614
Microsporidia are obligate intracellular opportunistic pathogens that infect a wide range of hosts. Human microsporidiosis is often attributed to Encephalitozoon species and is characterized by diarrhea and disseminated infections in the immunocompromised, particularly AIDS patients. Current hypotheses regarding microsporidial infection in humans make no concession for spore attachment to host cells, a process observed in vitro. Our previous studies show that E. intestinalis binds sulfated glycans, particularly heparin, on the host cell surface and that adherence to the host cell directly influences infection. The goal of this study is to identify and characterize microsporidia proteins that participate in this adherence process. In a host cell-binding assay, a single 40kDa E. cuniculi protein was isolated based on its ability to bind the host cell surface. This protein was identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry and database analysis from an excised Coomassie stained SDS polyacrylamide gel band. Analysis of the candidate protein sequence revealed three heparin-binding motifs and an RGD-like cell adhesion motif. The candidate protein was cloned and recombinantly expressed in E. coli for antibody production. A spore adherence assay was used to determine if the recombinant protein and polyclonal antisera influence E. cuniculi adherence to the host cell surface. The recombinant protein specifically inhibited spore adherence to host cells suggesting that the 40kDa E. cuniculi protein is involved in the spore adherence process. Spore adherence assays with the polyclonal antisera are currently underway. Ultimately, this research may provide a novel protein target for microsporidiosis therapeutics.
Carrie E. Jolly and James R. Hayman. Department of Microbiology, James H. Quillen College of Medicine, Johnson City TN.
The microsporidia, Encephalitozoon cuniculi and E. intestinalis, are spore-forming obligate intracellular protists which are increasingly being recognized as a cause of severe diarrhea and systemic infections in immunocompromised individuals. Very little is understood concerning the host-parasite interaction of microsporidiosis. In order to identify E. cuniculi spore proteins possibly involved in pathogenesis, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Two of the spore proteins identified by this method were heat shock protein 70 (HSP70)-related proteins, termed B1 and C1. Sequence analysis indicates that the C1 HSP70-related protein has an endoplasmic reticulum targeting signal while the B1 HSP70-related protein has neither a mitochondrial nor an endoplasmic reticulum targeting signal. The C1 HSP70-related protein was cloned and recombinantly expressed in E. coli. Antisera were generated against the recombinant C1 HSP70-related protein and specificity was demonstrated by Western analysis. Immuno-gold labeled transmission electron microscopy shows that the C1 HSP70-related protein is likely located in the cytoplasm of E. cuniculi. Sequence comparisons with previously isolated E. intestinalis cDNA clones have identified the E. intestinalis homologues of the B1 and C1 HSP70-related proteins. Since previous research indicates that some HSPs serve as immunodominant antigens during infectious disease, future studies in our laboratory will focus on determining the immunogenic fate of these HSP70-related proteins during infection with E. cuniculi and E. intestinalis.
THE DUAL ACTIVITIES OF ZMPSTE24 IN PRELAMIN A PROCESSING
Douglas Corrigan, Michael Sinensky, and Antonio Rusinol, Department of Biochemistry and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614
The transmembrane protease Zmpste24 is required for the processing of prelamin A to Lamin A, a process that involves two separate endoproteolytic cuts at the carboxy-terminus. Many disease states are associated with abnormal Lamin A function (due to either point mutations in Lamin A and/or Zmpste24 deficiency), such as Hutchinson–Gilford progeria syndrome (HGPS), muscular dystrophy, mandibuloacral dysplasia, dilated cardiomyopathy, and familial partial lipodystrophy. Our lab has just recently reported the first in-vitro evidence demonstrating that Zmpste24 has the ability to endoproteolytically process prelamin A at both sites. Our lab has previously ascribed this second activity to a chymotrypsin like protease. However, Zmpste24 is well known to have the canonical HEXXH domain, characteristic of a zinc metalloproteinase. Our most recent experiments on Zmpste24 demonstrate that inactivating the HEXXH domain by site directed mutagenesis results in a loss of the first endoproteolysis reaction. However, in this mutant the second activity is retained. Furthermore, we demonstrate in-vitro, that this second activity is sensitive to the chymotrypsin inhibitor TPCK and that purified Zmpste24 can be affinity labeled with FFCK, a fluorescent analogue to TPCK. Taken together, these experiments suggest that Zmpste24 contains a second, yet identified, active site with a chymotrypsin like catalytic mechanism. This work has been supported under NIH grant R01GM059578.