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Physiological and non-physiological regulation of indoleamine-2,3-dioxygenase
Title
Physiological and non-physiological regulation of indoleamine-2,3-dioxygenase.
Creator
Sempertegui Plaza, Tito S., Terentis, Andrew C., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry, Florida Atlantic University
Abstract/Description
The heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyses L-tryptophan (LTrp) oxidation along the kynurenine pathway and is a key regulator of the mammalian immune system. It’s unknown if the enzyme is under redox control and the use of new potent inhibitors of IDO represents a novel therapeutic approach.
Date Issued
2013
PURL
http://purl.flvc.org/fau/fd/FA00004256
Format
Document (PDF)
Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy
Title
Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy.
Creator
Cosme, Patrick Jason, Terentis, Andrew C., Florida Atlantic University, Charles E Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs,...
Show moreCell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies
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Date Issued
2016
PURL
http://purl.flvc.org/fau/fd/FA00004756
Subject Headings
Peptides--Analysis, Peptides--Therapeutic use, Peptides--Physiological transport, Cellular signal transduction, Raman spectroscopy, Infrared spectroscopy
Format
Document (PDF)
Raman micro-spectroscopy and multivariate analysis for the differentiation of skin cancers
Title
Raman micro-spectroscopy and multivariate analysis for the differentiation of skin cancers.
Creator
Fox, Sara A., Terentis, Andrew C., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Skin cancer is a growing health concern worldwide. In this work, confocal Raman micro-spectroscopy (CRM) was combined with two types of multivariate analysis, principal component analysis (PCA) and linear discriminant analysis (LDA), to accurately differentiate between skin cancer and normal skin. CRM was employed to study three distinct intracellular regions – cytoplasm, nucleoplasm, and nucleolus – within human metastatic melanoma (SK) and skin fibroblast (BJ) cells. PCA/LDA was 92-98%...
Show moreSkin cancer is a growing health concern worldwide. In this work, confocal Raman micro-spectroscopy (CRM) was combined with two types of multivariate analysis, principal component analysis (PCA) and linear discriminant analysis (LDA), to accurately differentiate between skin cancer and normal skin. CRM was employed to study three distinct intracellular regions – cytoplasm, nucleoplasm, and nucleolus – within human metastatic melanoma (SK) and skin fibroblast (BJ) cells. PCA/LDA was 92-98% successful in discriminating BJ from SK cells, with higher RNA identified in the nucleoli of BJ cells and higher lipids and collagen identified in the cytoplasm of SK cells. CRM measurements were also done on SCC, BCC, and normal skin tissue samples to determine the feasibility of combining Raman spectroscopy with CO2 ablation. Differentiation with PCA was possible between normal and SCC tissue that had been ablated, with 78% correct identification when non-ablated and 92% when ablated.
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Date Issued
2013
PURL
http://purl.flvc.org/fau/fd/FA00004247
Format
Document (PDF)
Raman and surface-enhanced raman spectroscopy of G-quadruplexes
Title
Raman and surface-enhanced raman spectroscopy of G-quadruplexes.
Creator
Friedman, Samantha, Terentis, Andrew C., Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
G-quadruplexes (G4s) are nucleic acid structures formed from π-stacked planar sets of four Hoogsteen hydrogen bonded guanine bases. G4s emerged as potential therapeutic targets based on their ability to modulate gene expression and inhibit the ability of telomerase to elongate chromosomal telomeres. Raman spectroscopy, polarized Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), and other optical spectroscopic techniques were used to characterize the G4s formed by four different...
Show moreG-quadruplexes (G4s) are nucleic acid structures formed from π-stacked planar sets of four Hoogsteen hydrogen bonded guanine bases. G4s emerged as potential therapeutic targets based on their ability to modulate gene expression and inhibit the ability of telomerase to elongate chromosomal telomeres. Raman spectroscopy, polarized Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), and other optical spectroscopic techniques were used to characterize the G4s formed by four different DNA sequences: human telomeric (HT), thrombin-binding aptamer (TBA), nuclease hypersensitive element III1 region of the c- Myc gene promoter (Myc), and a single loop-isomer of Myc (MycL1).
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Date Issued
2015
PURL
http://purl.flvc.org/fau/fd/FA00004370, http://purl.flvc.org/fau/fd/FA00004370
Subject Headings
Nucleic acids, Binding sites (Biochemistry), Biochemical genetics, Raman spectroscopy, Raman effect, Surface enhanced, Spectroscopic imaging, Spectrum analysis
Format
Document (PDF)
MECHANISTIC STUDIES OF THE PLP-DEPENDENT ENZYME, METHIONINE GAMMA-LYASE AND THE HEME-DEPENDENT ENZYME, INDOLEAMINE 2,3-DIOXYGENASE
Title
MECHANISTIC STUDIES OF THE PLP-DEPENDENT ENZYME, METHIONINE GAMMA-LYASE AND THE HEME-DEPENDENT ENZYME, INDOLEAMINE 2,3-DIOXYGENASE.
Creator
Foo, Timothy, Terentis, Andrew C., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description
Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density...
Show moreMethionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density functional theory (DFT) data for the γ-elimination reaction of L-Met and several other substrate analogues by MGL from P. gingivalis is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the γ-cleavage reaction of the enamine intermediate was identified for various substrates. Considering these data, we propose a catalytic mechanism for MGL catalysis, whereby the γ-cleavage step is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity. PLP-dependent enzymes are present in all organisms and account for ~4% of all discovered enzyme activities. They all possess a common external aldimine and quinonoid intermediate at the start of their reaction pathway but likely diverge thereafter into different reaction pathways based on their substrate(s) and active site environment. To yield insight into the divergent reactivity of the quinonoid intermediate, DFT calculations of several PLP-dependent enzymes with unique reactivity revealed that the charge distribution is modulated on Cα and C4’, which allows for the regioselectivity of the quinonoid intermediate. The mammalian heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Since IDO1’s catalytic mechanism remains uncertain, kinetic data and DFT data for the dioxygenation reaction of L-Trp and several other substrate analogues by human IDO1 is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the C2-O cleavage reaction of the L-Trp-epoxide intermediate was identified for various substrates. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating IDO1 activity and yields insight into heme-dependent dioxygenation chemistry.
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Date Issued
2021
PURL
http://purl.flvc.org/fau/fd/FA00013870
Subject Headings
Lyases, Enzymes
Format
Document (PDF)