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Article: alpha- and beta-monosaccharide transport in human erythrocytes.

Leitch, Jeffry M / Carruthers, Anthony

American journal of physiology. Cell physiology

2008 Volume 296, Issue 1, Page(s) C151–61

Abstract: ... This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM ...

Abstract	Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974-C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of beta- and alpha-sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents alpha-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported beta-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of alpha- and beta-sugar uptake demonstrates that erythrocytes transport alpha- and beta-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport beta-sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.
MeSH term(s)	3-O-Methylglucose/metabolism ; Adenosine Triphosphate/metabolism ; Biological Transport ; Carbohydrate Epimerases/metabolism ; Cell Shape ; Cytochalasin B/pharmacology ; Deoxyglucose/metabolism ; Erythrocytes/drug effects ; Erythrocytes/metabolism ; Glucose/metabolism ; Glucose Transporter Type 1/metabolism ; Humans ; Kinetics ; Maltose/metabolism ; Mannose/metabolism ; Models, Biological ; Monosaccharides/metabolism ; Phloretin/pharmacology
Chemical Substances	Glucose Transporter Type 1 ; Monosaccharides ; SLC2A1 protein, human ; 3-O-Methylglucose (146-72-5) ; Cytochalasin B (3CHI920QS7) ; Maltose (69-79-4) ; Adenosine Triphosphate (8L70Q75FXE) ; Deoxyglucose (9G2MP84A8W) ; Carbohydrate Epimerases (EC 5.1.3.-) ; aldose 1-epimerase (EC 5.1.3.3) ; Glucose (IY9XDZ35W2) ; Mannose (PHA4727WTP) ; Phloretin (S5J5OE47MK)
Language	English
Publishing date	2008-11-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00359.2008
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Article: ATP-dependent sugar transport complexity in human erythrocytes.

Leitch, Jeffry M / Carruthers, Anthony

American journal of physiology. Cell physiology

2006 Volume 292, Issue 2, Page(s) C974–86

Abstract: ... anomers in which V(max)/apparent K(m) for beta-3MG exchange transport is 19-fold greater than V(max ... apparent K(m) for alpha-3MG transport. ...

Abstract	Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar](intracellular) = [sugar](extracellular), where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of </=20 microM). Immunofluorescence-activated cell sorting analysis shows that biphasic exchange does not result from heterogeneity in cell size or GLUT1 content. Nucleoside transporter-mediated uridine exchange proceeds as rapidly as 3MG exchange but is monoexponential regardless of cytoplasmic [ATP]. This eliminates cellular heterogeneity or an ATP-dependent, nonspecific intracellular diffusion barrier as causes of biphasic exchange. Red cell ghost 3MG and uridine equilibrium volumes (130 fl) are unaffected by ATP. GLUT1 intrinsic activity is unchanged during rapid and slow phases of 3MG exchange. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional), or chemical (isomerization) barrier to equilibrate with cell water. Partial transport inhibition with the use of cytochalasin B or maltose depresses both rapid and slow phases of transport, thereby eliminating the physical barrier hypothesis. We propose that biphasic 3MG transport results from ATP-dependent, differential transport of 3MG anomers in which V(max)/apparent K(<mark>m) for beta-3MG exchange transport is 19-fold greater than V(max)/apparent K(m) for alpha-3MG transport.
MeSH term(s)	3-O-Methylglucose/metabolism ; Adenosine Triphosphate/physiology ; Biological Transport ; Cell Size ; Cytoplasm/metabolism ; Equilibrative Nucleoside Transporter 1/physiology ; Erythrocyte Membrane/metabolism ; Erythrocytes/metabolism ; Erythrocytes/ultrastructure ; Glucose/metabolism ; Glucose Transporter Type 1/metabolism ; Humans ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Models, Biological ; Uridine/metabolism
Chemical Substances	Equilibrative Nucleoside Transporter 1 ; Glucose Transporter Type 1 ; SLC2A1 protein, human ; 3-O-Methylglucose (146-72-5) ; Adenosine Triphosphate (8L70Q75FXE) ; Glucose (IY9XDZ35W2) ; Uridine (WHI7HQ7H85)
Language	English
Publishing date	2006-08-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00335.2006
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Article ; Online: The right to choose: multiple pathways for activating copper,zinc superoxide dismutase.

Leitch, Jeffry M / Yick, Priscilla J / Culotta, Valeria C

The Journal of biological chemistry

2009 Volume 284, Issue 37, Page(s) 24679–24683

Abstract: Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many ... ...

Abstract	Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the "activation" of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism's lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology.
MeSH term(s)	Animals ; Disulfides/chemistry ; Evolution, Molecular ; Humans ; Mitochondria/enzymology ; Models, Biological ; Models, Molecular ; Mutation ; Oxygen/chemistry ; Plants/genetics ; Saccharomyces cerevisiae/genetics ; Signal Transduction ; Superoxide Dismutase/chemistry ; Superoxide Dismutase/genetics ; Superoxide Dismutase/metabolism
Chemical Substances	Disulfides ; Superoxide Dismutase (EC 1.15.1.1) ; Oxygen (S88TT14065)
Language	English
Publishing date	2009-07-08
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.R109.040410
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Article ; Online: Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system.

Gross, Dominik P / Burgard, Caroline A / Reddehase, Silvia / Leitch, Jeffry M / Culotta, Valeria C / Hell, Kai

Molecular biology of the cell

2011 Volume 22, Issue 20, Page(s) 3758–3767

Abstract: The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/ ... ...

Abstract	The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.
MeSH term(s)	Cysteine/chemistry ; Cysteine/metabolism ; Cytosol/metabolism ; Disulfides/metabolism ; Gene Expression Regulation, Fungal ; Mitochondria/genetics ; Mitochondria/metabolism ; Mitochondrial Membrane Transport Proteins/genetics ; Mitochondrial Membrane Transport Proteins/metabolism ; Mitochondrial Membranes/metabolism ; Mitochondrial Proteins/genetics ; Mitochondrial Proteins/metabolism ; Models, Molecular ; Molecular Chaperones/chemistry ; Molecular Chaperones/genetics ; Molecular Chaperones/metabolism ; Mutation ; Oxidation-Reduction ; Oxidoreductases Acting on Sulfur Group Donors/genetics ; Oxidoreductases Acting on Sulfur Group Donors/metabolism ; Plasmids ; Protein Folding ; Protein Interaction Domains and Motifs ; Protein Transport/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Signal Transduction/genetics ; Superoxide Dismutase/genetics ; Superoxide Dismutase/metabolism ; Superoxide Dismutase-1 ; Transduction, Genetic
Chemical Substances	CCS1 protein, S cerevisiae ; Disulfides ; MIA40 protein, S cerevisiae ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Proteins ; Molecular Chaperones ; Saccharomyces cerevisiae Proteins ; Superoxide Dismutase (EC 1.15.1.1) ; Superoxide Dismutase-1 (EC 1.15.1.1) ; Oxidoreductases Acting on Sulfur Group Donors (EC 1.8.-) ; ERV1 protein, S cerevisiae (EC 1.8.3.2) ; Cysteine (K848JZ4886)
Language	English
Publishing date	2011-08-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1098979-1
ISSN	1939-4586 ; 1059-1524
ISSN (online)	1939-4586
ISSN	1059-1524
DOI	10.1091/mbc.E11-04-0296
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Article ; Online: Antigen-Based Testing but Not Real-Time Polymerase Chain Reaction Correlates With Severe Acute Respiratory Syndrome Coronavirus 2 Viral Culture.

Pekosz, Andrew / Parvu, Valentin / Li, Maggie / Andrews, Jeffrey C / Manabe, Yukari C / Kodsi, Salma / Gary, Devin S / Roger-Dalbert, Celine / Leitch, Jeffry / Cooper, Charles K

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

2021 Volume 73, Issue 9, Page(s) e2861–e2866

Abstract: Background: Individuals can test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by molecular assays following the resolution of their clinical disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to ... ...

Abstract	Background: Individuals can test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by molecular assays following the resolution of their clinical disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to be positive early in the disease course, when there is an increased likelihood of high levels of infectious virus. Methods: Upper respiratory specimens from 251 participants with coronavirus disease 2019 symptoms (≤7 days from symptom onset) were prospectively collected and tested with a lateral flow antigen test and a real-time polymerase chain reaction (rt-PCR) assay for detection of SARS-CoV-2. Specimens from a subset of the study specimens were utilized to determine the presence of infectious virus in the VeroE6TMPRSS2 cell culture model. Results: The antigen test demonstrated a higher positive predictive value (90%) than rt-PCR (70%) when compared to culture-positive results. The positive percentage agreement for detection of infectious virus for the antigen test was similar to rt-PCR when compared to culture results. Conclusions: The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture positivity represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. SARS-CoV-2 antigen testing can facilitate low-cost, scalable, and rapid time-to-result, while providing good risk determination of those who are likely harboring infectious virus, compared to rt-PCR.
MeSH term(s)	Antigens, Viral ; COVID-19 ; Humans ; Real-Time Polymerase Chain Reaction ; SARS-CoV-2 ; Sensitivity and Specificity
Chemical Substances	Antigens, Viral
Language	English
Publishing date	2021-01-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1099781-7
ISSN	1537-6591 ; 1058-4838
ISSN (online)	1537-6591
ISSN	1058-4838
DOI	10.1093/cid/ciaa1706
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Article: The Right to Choose: Multiple Pathways for Activating Copper,Zinc Superoxide Dismutase

Leitch, Jeffry M / Yick, Priscilla J / Culotta, Valeria C

Journal of biological chemistry. 2009 Sept. 11, v. 284, no. 37

2009

Abstract	Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme's biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the "activation" of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism's lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology.
Language	English
Dates of publication	2009-0911
Size	p. 24679-24683.
Publishing place	American Society for Biochemistry and Molecular Biology
Document type	Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
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Article ; Online: Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions.

Leitch, Jeffry M / Li, Cissy X / Baron, J Allen / Matthews, Lauren M / Cao, Xiaohang / Hart, P John / Culotta, Valeria C

Biochemistry

2012 Volume 51, Issue 2, Page(s) 677–685

Abstract: In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, ...

Abstract	In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakers' yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p β-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.
MeSH term(s)	Anaerobiosis/drug effects ; Animals ; Caenorhabditis elegans/drug effects ; Caenorhabditis elegans/enzymology ; Caenorhabditis elegans/growth & development ; Copper/metabolism ; Enzyme Activation/drug effects ; Models, Molecular ; Molecular Chaperones/metabolism ; Oxygen/pharmacology ; Phosphorylation/drug effects ; Protein Conformation ; Protein Processing, Post-Translational/drug effects ; Saccharomyces cerevisiae/drug effects ; Saccharomyces cerevisiae/enzymology ; Saccharomyces cerevisiae/growth & development ; Serine/metabolism ; Superoxide Dismutase/chemistry ; Superoxide Dismutase/metabolism ; Superoxide Dismutase-1 ; Zinc/metabolism
Chemical Substances	Molecular Chaperones ; Serine (452VLY9402) ; Copper (789U1901C5) ; Superoxide Dismutase (EC 1.15.1.1) ; Superoxide Dismutase-1 (EC 1.15.1.1) ; Zinc (J41CSQ7QDS) ; Oxygen (S88TT14065)
Language	English
Publishing date	2012-01-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/bi201353y
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Article: Post-Translational Modification of Cu/Zn Superoxide Dismutase under Anaerobic Conditions

Leitch, Jeffry M / Li Cissy X / Baron J. Allen / Matthews Lauren M / Cao Xiaohang / Hart P. John / Culotta Valeria C

Biochemistry. 2012 Jan. 17, v. 51, no. 2

2012

Abstract	In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakersâ yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p Î²-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.
Keywords	Caenorhabditis elegans ; Saccharomyces cerevisiae ; aerobic conditions ; anaerobic conditions ; enzyme activation ; hypoxia ; mass spectrometry ; mutants ; oxygen ; phosphorylation ; post-translational modification ; serine ; superoxide dismutase ; toxicity ; yeasts
Language	English
Dates of publication	2012-0117
Size	p. 677-685.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021%2Fbi201353y
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Article: Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Leitch, Jeffry M / Jensen, Laran T / Bouldin, Samantha D / Outten, Caryn E / Hart, P. John / Culotta, Valeria C

Journal of biological chemistry. 2009 Aug. 14, v. 284, no. 33

2009

Abstract: Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire ... ...

Abstract	Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.
Language	English
Dates of publication	2009-0814
Size	p. 21863-21871.
Publishing place	American Society for Biochemistry and Molecular Biology
Document type	Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
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Article: Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS.

Leitch, Jeffry M / Jensen, Laran T / Bouldin, Samantha D / Outten, Caryn E / Hart, P John / Culotta, Valeria C

The Journal of biological chemistry

2009 Volume 284, Issue 33, Page(s) 21863–21871

Abstract	Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.
MeSH term(s)	Animals ; Caenorhabditis elegans/metabolism ; Copper/chemistry ; Disulfides/chemistry ; Humans ; Kinetics ; Models, Biological ; Molecular Chaperones/chemistry ; Molecular Chaperones/metabolism ; Oxygen/chemistry ; Plasmids/metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/metabolism ; Superoxide Dismutase/chemistry ; Superoxide Dismutase/metabolism
Chemical Substances	CCS1 protein, S cerevisiae ; Disulfides ; Molecular Chaperones ; Saccharomyces cerevisiae Proteins ; Copper (789U1901C5) ; Superoxide Dismutase (EC 1.15.1.1) ; Oxygen (S88TT14065)
Language	English
Publishing date	2009-06-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M109.000489
Database	MEDical Literature Analysis and Retrieval System OnLINE

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