Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO
Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-05-01
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| Series: | Redox Biology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S221323172400096X |
| _version_ | 1827302678667984896 |
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| author | Pranjal Biswas Joseph Palazzo Simon Schlanger Dhanya Thamaraparambil Jayaram Sidra Islam Richard C. Page Dennis J. Stuehr |
| author_facet | Pranjal Biswas Joseph Palazzo Simon Schlanger Dhanya Thamaraparambil Jayaram Sidra Islam Richard C. Page Dennis J. Stuehr |
| author_sort | Pranjal Biswas |
| collection | DOAJ |
| description | Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology. |
| first_indexed | 2024-04-24T16:49:20Z |
| format | Article |
| id | doaj.art-33661509588b42689a85f3e9e0aeb44e |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-04-24T16:49:20Z |
| publishDate | 2024-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-33661509588b42689a85f3e9e0aeb44e2024-03-29T05:50:17ZengElsevierRedox Biology2213-23172024-05-0171103120Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NOPranjal Biswas0Joseph Palazzo1Simon Schlanger2Dhanya Thamaraparambil Jayaram3Sidra Islam4Richard C. Page5Dennis J. Stuehr6Department of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USADepartment of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USADepartment of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USADepartment of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USADepartment of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USADepartment of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USADepartment of Inflammation and Immunity, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH, 44195, USA; Corresponding author.Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.http://www.sciencedirect.com/science/article/pii/S221323172400096XHeme proteinMitochondriaHsp90Heme traffickingGAPDHIDO1 |
| spellingShingle | Pranjal Biswas Joseph Palazzo Simon Schlanger Dhanya Thamaraparambil Jayaram Sidra Islam Richard C. Page Dennis J. Stuehr Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO Redox Biology Heme protein Mitochondria Hsp90 Heme trafficking GAPDH IDO1 |
| title | Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO |
| title_full | Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO |
| title_fullStr | Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO |
| title_full_unstemmed | Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO |
| title_short | Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO |
| title_sort | visualizing mitochondrial heme flow through gapdh in living cells and its regulation by no |
| topic | Heme protein Mitochondria Hsp90 Heme trafficking GAPDH IDO1 |
| url | http://www.sciencedirect.com/science/article/pii/S221323172400096X |
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