Papers by Martha Stipanuk
Dr. Stipanuk's professional career has focused on the study of amino acid metabolism, particularl... more Dr. Stipanuk's professional career has focused on the study of amino acid metabolism, particularly the metabolism of the sulfur-containing amino acid cysteine. Her work has contributed to an understanding of the intermediary pathways of cysteine metabolism in mammalian cells and of the role of various tissues in cysteine metabolism, including glutathione synthesis and taurine production, within the whole body. The Stipanuk laboratory has played a major role in elucidating the physiological function, regulation, structure, and protein coenzyme formation of cysteine dioxygenase, an iron-dependent enzyme that catalyzes the first step in the cysteinesulfinate-dependent pathway of cysteine catabolism. Cysteine dioxygenase plays a crucial role in regulating cysteine levels, in promoting taurine biosynthesis, and in restricting the metabolism of cysteine through the intermediate hydrogen sulfide, preventing sulfide toxicity and facilitating hydrogen sulfide signaling. The regulation of cysteine dioxygenase abundance and activity state occurs specifically in response to cysteine levels. The Stipanuk laboratory is also interested in elucidating the mechanism(s) by which the concentrations of other amino acids are sensed by cells and how cells respond to changes in amino acid availability. Dr. Stipanuk has a long-standing interest in teaching and course development in the area of nutrient metabolism. She has developed a multi-authored advanced textbook entitled "Biochemical, Physiological and Molecular Aspects of Human Nutrition" published by Saunders/Elsevier with the 3rd edition released in 2012. Most recently, she has led efforts to develop and teach a graduate course on "Regulation of Macronutrient Metabolism." She also teaches a specialized graduate course on "Nutritional Regulation of Mammalian Protein Synthesis and Degradation." My current teaching is primarily at the graduate student level. In this context, I try My current teaching is primarily at the graduate student level. In this context, I try to focus classwork and assignments on topics relevant to major contemporary research questions in molecular nutrition and metabolism, incorporating use of the literature and development of conceptual mapping of topics to faciliate an understanding of multiple and complex pathways and physiological processes involved in particular research areas. In advising and mentoring both undergraduates and graduate students, I encourage students to pursue a range of areas of study and experiential learning relevant to their interests and to consider various possibilities for future study and careers.
Dr. Stipanuk's professional career has focused on the study of amino acid metabolism, particularl... more Dr. Stipanuk's professional career has focused on the study of amino acid metabolism, particularly the metabolism of the sulfur-containing amino acid cysteine. Her work has contributed to an understanding of the intermediary pathways of cysteine metabolism in mammalian cells and of the role of various tissues in cysteine metabolism within the whole body. The Stipanuk laboratory is currently investigating the molecular mechanisms involved in regulation of levels of two key regulatory enzymes of cysteine metabolism, cysteine dioxygenase and glutamate-cysteine ligase in response to dietary protein or amino acid levels. These enzymes play a crucial role not only in regulating cysteine levels but in regulating the synthesis of taurine, sulfate, glutathione, and hydrogen sulfide, compounds that are necessary for health. A genetic lack of adequate levels of functional cysteine dioxygenase has been shown to be an important factor in the development of many cases of rheumatoid arthritis. Because the regulation of these two enzymes occurs specifically in response to cysteine levels, the Stipanuk laboratory is also interested in elucidating the mechanism(s) by which the concentrations of cysteine and other amino acids are sensed by cells and how cells respond to changes in amino acid availability. Dr. Stipanuk has a long-standing interest in teaching and course development in the area of nutrient metabolism. She has developed a multi-authored advanced textbook entitled "Biochemical, Physiological and Molecular Aspects of Human Nutrition" published by Saunders/Elsevier with the 3rd edition expected to be released in spring 2012. Most recently, she has led efforts to develop and teach a graduate course on "Regulation of Macronutrient Metabolism." She also teaches a specialized graduate course on "Nutritional Regulation of Mammalian Protein Synthesis and Degradation.
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), May 1, 1986
In studies with rat hepatocytes, hypotaurine plus taurine production accounted for less than 5% o... more In studies with rat hepatocytes, hypotaurine plus taurine production accounted for less than 5% of the total amount of cysteine (CYS) catabolized, whereas more than 90% of the metabolized cysteinesulfinate (CSA) was converted to taurine plus hypotaurine. Similar studies have been carried out with kidney tubules isolated from fed rats and incubated with 2 mM (1-/sup 14/C)CYS or 25 mM (1-/sup 14/C)CSA at 37/sup 0/C for up to 40 min. The production of /sup 14/CO/sub 2/ from CSA (3.1 +/- 1.3 nmol/sup ./ min/sup -1//sup ./ mg dry wt/sup -1/) was equivalent to the accumulation of N in NH/sub 4//sup +/ plus glutamate. Substantial oxidation of CYS was observed (16 +/- 11 nmol CO/sub 2/ x min/sup -1/ x mg dry wt/sup -1/), but only 12% of the expected amount of N was recovered as NH/sub 4//sup +/ plus glutamate. Accumulation of hypotaurine plus taurine was equivalent to 20% of the observed rate of /sup 14/CO/sub 2/ production from CSA but accounted for only 2% of the observed rate of /sup 14/CO/sub 2/ production from CYS. Addition of unlabeled CSA to incubations with varying levels of CYS had no effect on production of /sup 14/CO/sub 2/. Addition of 2 mMmore » ..cap alpha..-ketoglutarate to the incubation mixtures resulted in an increased in /sup 14/CO/sub 2/ production from CSA to 290% of the control level but had no effect on CYS oxidation. In agreement with the authors findings for rat hepatocytes, these data suggest that most metabolism of CYS by the rat kidney tubule occurs by a CSA-independent pathway. However, in contrast to the metabolism of CSA almost entirely to taurine in the hepatocyte, kidney tubules appeared to metabolize CSA primarily by the transamination pathway.« less
Physiological Research, Oct 31, 2021
Increased plasma total cysteine (tCys) has been associated with obesity and metabolic syndrome in... more Increased plasma total cysteine (tCys) has been associated with obesity and metabolic syndrome in human and some animal studies but the underlying mechanisms remain unclear. In this study, we aimed at evaluating the effects of high cysteine diet administered to SHR-CRP transgenic rats, a model of metabolic syndrome and inflammation. SHR-CRP rats were fed either standard (3.2 g cystine/kg diet) or high cysteine diet (HCD, enriched with additional 4 g L-cysteine/kg diet). After 4 weeks, urine, plasma and tissue samples were collected and parameters of metabolic syndrome, sulfur metabolites and hepatic gene expression were evaluated. Rats on HCD exhibited similar body weights and weights of fat depots, reduced levels of serum insulin, and reduced oxidative stress in the liver. The HCD did not change concentrations of tCys in tissues and body fluids while taurine in tissues and body fluids, and urinary sulfate were significantly increased. In contrast, betaine levels were significantly reduced possibly compensating for taurine elevation.
Biochemical journal. Cellular aspects, Aug 15, 1982
The contribution of cystathionine y-lyase, cystathionine,f-synthase and cysteine amino- transfera... more The contribution of cystathionine y-lyase, cystathionine,f-synthase and cysteine amino- transferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydra- tion in rat liver and kidney was assessed with four different assay systems. Cyst- athionine y-lyase and cystathionine J-synthase were active when homogenates were incubated with 280mM-L-cysteine and 3 mM-pyridoxal 5'-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160mM-L-cysteine, 2mM-pyridoxal 5'-phosphate, 3mM-2-oxoglutarate and 3mM-dithiothreitol. At more- physiological concentrations of cysteine (2 mM) cystathionine y-lyase and cystathionine ,6-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine y-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO42or [35S]taurine formed from labelled dietary cysteine. The desulphhydration of cyst(e)ine may be cata- lysed by several enzymes present in mammalian tissues. These include cysteine aminotransferase (EC 2.6.1.3) in conjunction with 3-mercapto- pyruvate sulphurtransferase (EC 2.8.1.2), cyst- athionine y-lyase (EC 4.4.1.1) and cystathionine fisynthase (EC 4.2.1.22). The reactions catalysed by these enzymes are summarized in Scheme 1. The reactions may be important not only in cysteine catabolism but also as a source of metabolically active reduced sulphur. Other known pathways of cysteine degradation in animal tissues lead to release of sulphur in the 4+ or 6+ oxidation state. This highly oxidized sulphur is irreversibly lost to the pool of metabolically active reduced sulphur because of the absence of sulphateand sulphite-reducing systems in animal tissues . Cysteine participates in transamination reactions with various amino acceptors, and it is likely that several non-specific aminotransferases catalyse its transamination to 3-mercaptopyruvate Ubuka et al., ,b, 1978)). Aspartate aminotransferase catalyses the transamina- *tion of cysteine with 2-oxoglutarate in vitro and may also be active in vivo . The keto acid of cysteine is decomposed to inorganic sulphur and pyruvate by both desulphuration and'trans-sulphuration reactions (Meister et al.,
C-Reactive Protein and Cardiovascular Disease, edited by Drs Paul Ridker and Nader Rifai from the... more C-Reactive Protein and Cardiovascular Disease, edited by Drs Paul Ridker and Nader Rifai from the Harvard Medical School and with a foreword by Dr Eugene Braunwald, is a timely and interesting review of the prominent role played by inflammation in atherosclerosis. It focuses on the prototypic marker of inflammation in man-ie, C-reactive protein (CRP)-and its role as a novel marker of cardiovascular disease risk.
FASEB Journal (Federation of American Societies for Experimental Biology); (United States), Feb 26, 1990
To assess the extent to which low he patic y-cystathionase levels affect methionine flux to cyste... more To assess the extent to which low he patic y-cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of y-cystathionase activity with propargylglycine on the metabolism of L-[35S]methionine was determined in studies with freshly isolated rat hepatocytes. y-Cystathionase activity was inhibited 25%, 42%, 63% and 76% (maximal inhibition) by treatment with 2.5 umol/L, 0.01 mmol/L, 0.02 mmol/L and 2 mmol/L propargylglycine, respectively. Inhibition of y-cysta thionase activity with up to 0.02 mmol/L propar gylglycine had no statistically significant effect on pSjglutathione, [35SJsulfate or [3?S]cysteine forma tion from [35S]methionine. However, treatment of cells with 2 mmol/L propargylglycine markedly inhibited the metabolism of [35S]methionine to [35S]glutathione by 93%, to [35S)sulfate by 88% and to [3%Jcysteine by 89%; [35S]cystathionine accumulation in these incuba tion systems was 60 times control. Hepatic y-cys tathionase activity in premature infants has been reported to be about 23% of mature levels (Zlotkin and Anderson, 1982; Pediatr. Res. 16: 65-68); this level of Y-cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway. No evi dence for cysteine synthesis from serine and sulfide, which can be catalyzed by cystathionine ß-synthase, or for methionine metabolism by an S-adenosyImethionine-independent pathway was obtained.
The Journal of Nutrition, 2020
ABSTRACTMetabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facili... more ABSTRACTMetabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facilitated by the expression of methionine adenosyltransferase (MAT) I/III and glycine N-methyltransferase (GNMT) in liver, and a lack of either enzyme results in hypermethioninemia despite normal concentrations of MATII and methyltransferases other than GNMT. The further metabolism of Hcy by the transsulfuration pathway is facilitated by activation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) as well as the relatively high KM of CBS for Hcy. Transmethylation plus transsulfuration effects catabolism of the Met molecule along with transfer of the sulfur atom of Met to serine to synthesize cysteine (Cys). Oxidation and excretion of Met sulfur depend upon Cys catabolism and sulfur oxidation pathways. Excess Cys is oxidized by cysteine dioxygenase 1 (CDO1) and further metabolized to taurine or sulfate. Some Cys is normally metabolized by desulfhydration pathways, and the hydrog...
Encyclopedia of Inorganic and Bioinorganic Chemistry, 2015
Cysteine dioxygenase (CDO) is a mononuclear nonheme Fe(II) enzyme that catalyzes the oxidation of... more Cysteine dioxygenase (CDO) is a mononuclear nonheme Fe(II) enzyme that catalyzes the oxidation of l-cysteine to l-cysteinesulfinic acid (CSA) by addition of both atoms of molecular oxygen to the cysteine sulfur. Mammalian CDO is highly expressed in the liver, the pancreas, the adipose tissue, the kidney and the lungs, and the CSA produced is further converted to either taurine or sulfate plus pyruvate. To maintain cysteine levels within the narrow range required for health, mammalian CDO is regulated by the modulation of enzyme turnover and through formation of a Cys93-Tyr157 crosslink that increases its activity over 10-fold. Imbalances in cysteine metabolism have been observed in several neurological disorders, and CDO has been identified as a tumor suppressor. Bacterial CDOs also exist, as do a variety of related thiol dioxygenases that have been less well studied. High-resolution crystal structures of CDO reveal iron coordination by three histidines. Cysteine coordinates the iron by its amino and thiolate groups, leaving a sixth coordination site open for dioxygen. A cysteine persulfenate has been trapped in the active site, but despite substantial spectroscopic studies and theoretical calculations, the details of the mechanism are still a matter of debate and it is uncertain if this is an intermediate. 3D Structure Structure of rat CDO with cysteine persulfenate (or cysteine persulfenic acid) bound based on PDB code 4ieu1. The cupin fold of CDO is shown with the secondary structures labeled; the iron at the center of the β-barrel is shown as a black sphere. The Cys–Tyr crosslink is shown, along with the active site Arg, the iron coordinating His residues and the bound cysteine persulfenate. Figure generated using the PyMOL Molecular Graphics System, Version 1.5.0.4 Schrodinger, LLC.2 Keywords: thiol oxidation; sulfur metabolism; nonheme iron; cysteine; dioxygenase; cysteinesulfinic acid; taurine; sulfate
Intructor's manual for biochemical and physiological aspects of human nutrition , Intructor&#... more Intructor's manual for biochemical and physiological aspects of human nutrition , Intructor's manual for biochemical and physiological aspects of human nutrition , کتابخانه مرکزی دانشگاه علوم پزشکی تهران
The FASEB Journal, Apr 1, 2013
American Journal of Physiology-endocrinology and Metabolism, May 1, 1993
The metabolism of cysteine and related compounds was investigated in the isolated perfused hindqu... more The metabolism of cysteine and related compounds was investigated in the isolated perfused hindquarter of the rat. An erythrocyte-based perfusion medium was used; use of a perfluorochemical emulsion, FC-43, resulted in apparent chemical oxidation of cysteine, whereas bovine erythrocytes did not appear to contribute significantly to the metabolism of cysteine. Rat skeletal muscle perfused with L-[35S]cysteine, L-[35S]cystine, L-[35S]cysteinesulfinate, or L-2-oxo-[35S]thiazolidine-4-carboxylate (OTC) for 2 h produced [35S]sulfate and [35S]taurine. In all cases, the partitioning of cysteine or cysteinesulfinate between metabolism to taurine and sulfate was similar, suggesting that cysteine metabolism in hindquarter may occur via formation and catabolism of cysteinesulfinate by either cysteinesulfinate decarboxylase or aspartate (cysteine-sulfinate) aminotransferase. However, the activity of cysteine dioxygenase was extremely low, suggesting that the conversion of cysteine to cysteinesulfinate may have been non-enzymatic.
Advances in Experimental Medicine and Biology, 1994
Protein Expression and Purification, May 1, 2006
The expression of soluble recombinant transglutaminase (TGase) has proven to be a challenge for m... more The expression of soluble recombinant transglutaminase (TGase) has proven to be a challenge for many research groups. Herein, we report a complementary method for the expression, in BL21(DE3) Escherichia coli, of recombinant human tissue transglutaminase (hTG2) whose solubility is enhanced through N-terminal fusion to glutathione S-transferase (GST). Moreover, we report the cleavage of the GST tag using PreScission™ Protease (PSP) and purification of hTG2 in its untagged form, distinctively suitable for subsequent studies of its remarkable conformational equilibrium. The effects of co-solvents and storage conditions on stability of purified hTG2 are also reported. Furthermore, we demonstrate for the first time the use of a convenient chromogenic assay to measure the activity of the human enzyme. The utility of this assay was demonstrated in the measurement of the kinetic parameters of a wide variety of substrates and inhibitors of both hTG2 and the extensively studied guinea pig liver TGase. Finally, comparison of these results provides further evidence for the functional similarity of the two enzymes.
Scientific Reports, May 24, 2018
Although amino acids are known regulators of translation, the unique contributions of specific am... more Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation. The effects of amino acid availability on the regulation of protein synthesis are believed to be mediated predominantly through the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which appears to be especially sensitive to leucine availability 1,2 , and through regulation of the phosphorylation status of the alpha subunit of eukaryotic initiation factor 2 (eIF2) 3. A major downstream target of mTORC1 signaling is the eukaryotic initiation factor 4E binding proteins (4EBP1/2). Most research has been conducted on 4EBP1, but 4EBP2 appears to function similarly. 4EBP competes with eIF4G for binding to eIF4E 4-7. When eIF4E is bound to 4EBP, eIF4E recognizes the 5′ cap but cannot recruit eIF4G 4-7. Effects of mTORC1 signaling on translation initiation are usually attributed to its phosphorylation of 4EBP 4,8-12. Under nutrient-sufficient conditions mTORC1 phosphorylates 4EBP at several sites, which inhibits 4EBP binding to eIF4E and promotes cap-dependent translation. Conversely, mTORC1 is inhibited under conditions such as low insulin or low amino acids, and 4EBP becomes hypophosphorylated and bound to eIF4E, thus inhibiting translation 4,8-12. Leucine is particularly effective in activating mTORC1 2,13,14 , and both Sestrin2 1 and leucyl-tRNA synthetase 15 have been identified as cytosolic leucine sensors that promote mTORC1 localization to the lysosomal membrane and activation of mTORC1 kinase activity when they are in their leucine-bound forms. Translation initiation is also regulated in response to amino acids by phosphorylation of the alpha subunit of eIF2. When an essential amino acid is limiting, its tRNA(s) cannot be fully aminoacylated and the concentration(s) of the non-aminoacylated ("uncharged") tRNA(s) increase(s) 16. Uncharged tRNAs bind to the histidyl-tRNA synthetase−related regulatory domain of GCN2 (i.e., eIF2α kinase 4), causing its activation 17-21. Active GCN2 then phosphorylates the alpha subunit of eIF2 22-24. eIF2 functions in its GTP-loaded form to form the 43S preinitiation complex (PIC) with initiator methionine tRNA (Met-tRNA i Met), the 40S ribosomal subunit, eIF3, eIF1 and eIF1A. The engagement of the PIC with a start codon results in hydrolysis of eIF2-GTP to
The FASEB Journal, Apr 1, 2013
The FASEB Journal, Apr 1, 2013
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Papers by Martha Stipanuk