Poster Preparation more costly and time-consuming Opportunity to interact with audience Possible to develop collaborations with co-presenters Considered less prestigious than oral presentation at some conferences Oral Quick preparation at no cost Little interaction with audience outside of 5 min Q&A Little time to discuss project with co-presenters Oral presentations are sometimes chosen as the best in that area Poster vs Oral Presentation
Text on poster must be legible from a viewing distance of 6 feet. • For section headings (e.g., Introduction), use boldface, maybe about 36-42 point. For supporting text (e.g., text within each section & figure captions), use about 24-28 point (boldface, if appropriate). • In general, use font sizes proportional to importance: • largest type - Title • next largest type - Section headings • medium type - Supporting material • smallest type - Details • If you have your poster made at MUSM, Elizabeth Hyde in Illustration will properly size the text for you from the Word and PowerPoint files that you send her by email: • HYDE_EM@Mercer.edu
TITLE, AUTHORS, INSTITUTIONS Differences in the Kinetics of Substrate Utilization by Human Type 1 and Type 2 3β-Hydroxy-steroid Dehydrogenase (3β-HSD) are Structurally Characterized by the Type 1 Chimera, H156Y. James L. Thomas1*, J. Ian Mason2, Stacey Brandt1 and Wendy Norris1 1Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA, 31207 and 2Department of Reproductive & Developmental Sciences (Clinical Biochemistry), University of Edinburgh, Edinburgh, Scotland, United Kingdom.
ABSTRACT Two distinct genes encode the 93% homologous human type 1 (placenta, mammary gland, prostate) and type 2 (gonads, adrenals) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/isomerase). Our mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif that is present in the primary structure of the human type 13β-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has been created to produce a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The wild-type 1 and 2 enzymes plus these three mutants were expressed and purified to homogeneity. Michaelis-Menten constants of substrate conversion were measured for each purified enzyme. The wild-type 2 Km value for dehydroepiandrosterone is 12.7-fold greater and the Vmax is 1.9-fold greater than those values measured for the wild-type 13β-HSD activity. The H156Y type 1 mutant has kinetic constants for 3β-HSD activity that are very similar to those of the wild-type 2 enzyme. In contrast, the kinetic values measured for the isomerization of 5-androstene-3,17-dione by the H156Y type 1 mutant are almost identical to those measured for the wild-type 1 isomerase. The Y154F and K158Q type 1 mutants exhibit no 3β-HSD activity and have Km values that are similar to the wild-type 1 isomerase values. Thus, a structural basis for the difference in the substrate kinetics of the wild-type 1 and 2 3β-HSD and the critical residues for the3β-HSD reaction have been identified. This significant advance in our understanding of the structure/function of human type 1 and 2 3β-HSD/isomerase may lead to the selective inhibition of the type 1 enzyme not only in placenta to control the onset of labor but also in prostate and breast tumors to slow their hormone-dependent growth.(Supported by NIH grant HD20055, JLT)
Clinical Abstract: OBJECTIVES: (1) Characterize serum (S) and urinary (U) steroid metabolites in complete CYP17 deficiency (cCYP17D); (2) analyze the relative 17α-hydroxylase (17OH) and 17,20-lyase (17,20L) activities in vivo; and (3) comparedata from the two most prevalent mutations in Brazil. SUBJECTS AND METHODS: 20 genotyped cCYP17D patients from a previously reported cohort were homozygous for W406R or R362C; 11 controls were CYP17 wild types (WT). WT and cCYP17D patients had S and U samples drawn to measure: cortisol (F), corticosterone (B), deoxycorticosterone (DOC), 18OH-B, 18OH-DOC, and 17OHP; and tetrahydro (TH)-B, THA, THDOC, THF+5α-THF, TH-cortisone, androsterone, etiocholanolone, 5-pregnenediol, 17OH-pregnenolone and pregnanetriol. RESULTS: Compared to WT, cCYP17D patients had marked elevations of B, DOC, 18OH-B and 18OH-DOC, whereas 17OHP, F and adrenal androgens (AA) were reduced; U steroids parallel S findings. Metabolite ratios revealed that both 17OH and 17,20L activities were impaired in cCYP17D. There were nodifferences between W406R andR362C mutations. CONCLUSIONS: cCYP17D patients show parallel overproduction/overexcretion of 17-deoxysteroids, and marked reduction of F and AA. In addition to 17OH, 17,20-L activity was also impaired in cCYP17D. W406 and R362C mutations disclose similar Sand U patterns.
Purposes or INTRODUCTION • Describe the purposes of the study • Three main points • Significance of study
PURPOSES • Produce and express the human wild-type 1 (placenta, breast tumors, prostate tumors) and wild-type 2 (adrenals, gonads) 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD/isomerase) as well as potentially critical mutant forms of the type 1 enzyme. • Purify each enzyme and compare the substrate and cofactor kinetics of the 3β-HSD and isomerase activities of the wild-type 1 and wild-type 2 enzymes as well as the mutants of the type 1 enzyme. • The H156Y mutation creates the type 2 catalytic motif (Y154-P-Y156-S-K158) in the type 1 enzyme (Y154-P-H156-S-K158). The Y-X-X-S-K motif is responsible for the enzyme activity of several hydroxysteroid dehydrogenases. This structural difference may be the basis for the selective inhibition of human type 1 3β-HSD.
INTRODUCTION The human type 1 isoform (breast tumors, placenta) of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD1) is a key enzyme in the steroidogenic pathway leading to the production of estradiol. Human 3β-HSD2 is selectively expressed in the adrenal glands and ovaries in the female.The goals of this project are to evaluate whether Arg195 in 3β-HSD1 vs Pro195 in 3β-HSD2 is a critical structural difference responsible for the inhibition of 3β-HSD1 by trilostane with 16-fold higher-affinity compared to 3β-HSD2 and to determine if a 16β-hydroxyl group on DHEA increases the affinity of 3β-HSD1, but not of 3β-HSD2, for substrates. Using our structural PDB file of human 3β-HSD1, docking of the inhibitor, trilostane, and substrates, DHEA and 16β-hydroxy-DHEA, with 3β-HSD1 has predicted that Arg195 in 3β-HSD1 interacts with the steroid 16,17-oxo groups and that Pro195 in 3β-HSD2 does not. To test this prediction, the R195P-1 mutant of 3β-HSD1 and the P195R-2 mutant of 3β-HSD2 have been created, expressed, purified and characterized kinetically.
SIGNIFICANCE Human 3β-hydroxysteroid dehydrogenase is a key enzyme in the biosynthesis of active steroid hormones which is expressed as two tissue-specific isoforms. Because 3β-HSD1 is expressed in placenta and breast tumors, and 3β-HSD2 is expressed in the adrenal glands, it may be possible to selectively inhibit 3β-HSD1 in placenta to control the timing of labor or in breast tumors to slow their growth without affecting the essential steroidogenic activity of adrenal 3β-HSD2. Note: Significance can be incorporated into the Introduction without having a separate section.
CYP17 Pregnenolone 17-OH-Pregnenolone DHEA HUMAN 3b-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE Type 1 3-HSD in placenta and hormone-sensitive tumors Type 2 3-HSD in adrenal cortex Progesterone 17b-HSD CYP19, 17b-HSD CYP21, CYP11B2 CYP21, CYP11B1 CYP19 17b-Estradiol Testosterone Cortisol Aldosterone Schematic showing significance
Figure 1. Subunit interface of human 3b-hydroxysteroid dehydrogenase showing interactions between key amino acids.
3b-HSD2 3b-HSD1 H156Y Y154F Y158Q Figure 3. Western blot of an SDS-polyacrylamide gel shows expression of the mutant enzymes by baculovirus. The Sf9 cell homogenates (1.5 μg each) contained Y154F, K158Q, or H156Y. Purified wild-type 1 and wild-type 2 3-HSD/isomerase (0.05 g each) served as controls.
Figure 3. Dixon analysis of the inhibition of MCF-7 3β-HSD1 and MCF-7 3β-HSD2 cell homogenates (0.3 mg) by epostane (0-0.75 mM, 3β-HSD1; 0-7.5 mM, 3β-HSD2).
Table 2. Comparison of inhibition constants of trilostane for purified human R195P-1, 3b-HSD1, P195R-2 and 3b-HSD2. Enzyme Trilostane Ki (M) 1 R195P-1 2.56 ± 0.22 3b-HSD1 0.10 ± 0.01 (C) P195R-2 0.19 ± 0.02 (C) 3b-HSD2 1.60 ± 0.10 1 For 3-HSD1 and P195R-2, the incubations contained sub-saturating concentrations of DHEA (4.0 μM or 8.0 M), NAD+ (0.2 mM), purified human 3-HSD type 1 enzyme (0.04 mg) and trilostane (0-1.0 M) in 0.02 M potassium phosphate buffer, pH 7.4. For 3β-HSD2 and R195P-1, similar incubations contained DHEA (8.0 μM or 20.0 μM) and trilostane (0-7.5 M). Dixon analysis (I versus 1/V) was used to determine the mode of inhibition and calculate the Ki values. (C) denotes a competitive mode of inhibition, and no notation indicates a noncompetitive mode of inhibition.
CONCLUSIONS • The Y154F and K158Q mutations abolished the 3β-HSD activity and significant isomerase activity was retained. Therefore, Tyr154 and Lys158 are essential for 3b-HSD activity and may be catalytic residues. • The H156Y mutation of the type 1 enzyme shifted the Michaelis-Menten substrate kinetic profile for 3β-HSD1 to that of the 3β-HSD2 (12-fold higher Km, 2-fold higher Vmax). • The H156vs Y156 structure/function relationship is responsible for the substantially higher affinity of type 1 3β-HSD for substrate and inhibitor steroids compared to the type 2 3β-HSD.
FORMAT OF POSTER • Single large poster • Separate Panels
Pros Cons Looks more professional (perhaps this is debatable?) Requires access to specialized printers (or Kinkos) Rolls easily for storage and travel (especially if laminated) Expensive to print (especially if you notice a typo!) Becoming increasingly popular and perhaps even expected More difficult to create More flexibility in arranging items (not boxed into small chunks of information) in terms of the overall picture Less flexibility in the long run (less likely to be usable for another conference if, for example, the poster boards at one meeting are horizontal but at another are vertical) Can easily make handouts identical to your poster Large Single Poster
Pros Cons Less expensive to print overall and easier to reprint section by section in case of typos. Can be prepared yourself without a special printer. Looks less professional if not well done Easier to transport because pieces fit in your briefcase (title-boards can be hinged together to fold compactly) Shorter shelf life than a laminated poster Easier to design; can even be arranged differently at meeting More flexible (easily used at other conferences; in different layouts) Multiple Small Panels
HANDOUT • 1-2 page handout in manila envelope attached to poster board. • Print the single large poster as an 8.5 x 11 inch sheet. • Gives viewers something to take home who may contact you later.