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Atomic-resolution monitoring of protein maturation in live human cells by NMR.

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR - Nat. Chem. Biol. (2013)

Bottom Line: We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1).We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms.Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

View Article: PubMed Central - PubMed

Affiliation: CERM, Magnetic Resonance Center, University of Florence, Florence, Italy. banci@cerm.unifi.it

ABSTRACT
We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

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Cu(II) addition to the culture medium induces Cu(I) binding to a fraction of cytoplasmic SOD1Histidine region of 1H NMR spectra were acquired on human cells expressing unlabelled SOD1: a, in Zn(II)-supplemented medium, after incubation with Cu(II); b, in Zn(II)-supplemented medium without incubation with Cu(II); c, in medium without added metals. d, 1H NMR spectrum of human cells co-expressing SOD1 and CCS in Zn(II)-supplemented medium, after incubation with Cu(II). Histidine protons unambiguously assigned to Cu(I),Zn-SOD1 species are indicated.
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Figure 2: Cu(II) addition to the culture medium induces Cu(I) binding to a fraction of cytoplasmic SOD1Histidine region of 1H NMR spectra were acquired on human cells expressing unlabelled SOD1: a, in Zn(II)-supplemented medium, after incubation with Cu(II); b, in Zn(II)-supplemented medium without incubation with Cu(II); c, in medium without added metals. d, 1H NMR spectrum of human cells co-expressing SOD1 and CCS in Zn(II)-supplemented medium, after incubation with Cu(II). Histidine protons unambiguously assigned to Cu(I),Zn-SOD1 species are indicated.

Mentions: Prokaryotes have simple mechanisms for copper uptake and excretion, while they are tightly regulated in eukaryotic cells16. Accordingly, copper added as Cu(II) salt to E. coli cells culture medium was reduced to Cu(I) and readily and stoichiometrically bound only in this redox state to recombinantly expressed SOD1, forming Cu(I),Zn-SOD1 (Supplementary Fig. 7a,b). Importantly, Cu(I) added to E. coli cells either as an acetonitrile or glutathione complex did not become available to E,Zn-SOD1 (Supplementary Fig. 7c). In-cell NMR spectra also showed that in E.coli the SOD1 intrasubunit disulfide bridge is oxidized in a sizable fraction (around 50%, Supplementary Fig. 7d). Unlike bacteria, copper entrance in eukaryotic cells and its delivery to copper-binding proteins require a number of steps, involving specific chaperones responsible of its intracellular trafficking17,18. Accordingly, only around 25% of the recombinant SOD1 protein incorporated copper, again in the Cu(I) state, when human cells were cultured in the presence of Cu(II) (Fig. 2a). The remaining SOD1 fraction contained only one zinc ion per subunit, as observed from the 1H histidine signals in the 1D 1H NMR spectrum (Fig. 2a-c). Additionally, the spectra of the 15N-Cys selectively labelled protein showed only ~20% SOD1 intrasubunit disulfide bond formation (Supplementary Fig. 8). Copper incorporation of SOD1 in eukaryotes is known to be dependent on the CCS protein19,20. Although a basal expression-level of the hCCS gene does occur during normal cell growth, it is likely that the amount of SOD1 produced in our experimental setup was too high to allow for complete copper insertion via the CCS-dependent pathway. However, a CCS-independent copper insertion pathway has also been reported for human SOD121,22 and might have contributed to the partial formation of Cu(I),Zn-SOD1 observed (Fig 2a).


Atomic-resolution monitoring of protein maturation in live human cells by NMR.

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR - Nat. Chem. Biol. (2013)

Cu(II) addition to the culture medium induces Cu(I) binding to a fraction of cytoplasmic SOD1Histidine region of 1H NMR spectra were acquired on human cells expressing unlabelled SOD1: a, in Zn(II)-supplemented medium, after incubation with Cu(II); b, in Zn(II)-supplemented medium without incubation with Cu(II); c, in medium without added metals. d, 1H NMR spectrum of human cells co-expressing SOD1 and CCS in Zn(II)-supplemented medium, after incubation with Cu(II). Histidine protons unambiguously assigned to Cu(I),Zn-SOD1 species are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4017183&req=5

Figure 2: Cu(II) addition to the culture medium induces Cu(I) binding to a fraction of cytoplasmic SOD1Histidine region of 1H NMR spectra were acquired on human cells expressing unlabelled SOD1: a, in Zn(II)-supplemented medium, after incubation with Cu(II); b, in Zn(II)-supplemented medium without incubation with Cu(II); c, in medium without added metals. d, 1H NMR spectrum of human cells co-expressing SOD1 and CCS in Zn(II)-supplemented medium, after incubation with Cu(II). Histidine protons unambiguously assigned to Cu(I),Zn-SOD1 species are indicated.
Mentions: Prokaryotes have simple mechanisms for copper uptake and excretion, while they are tightly regulated in eukaryotic cells16. Accordingly, copper added as Cu(II) salt to E. coli cells culture medium was reduced to Cu(I) and readily and stoichiometrically bound only in this redox state to recombinantly expressed SOD1, forming Cu(I),Zn-SOD1 (Supplementary Fig. 7a,b). Importantly, Cu(I) added to E. coli cells either as an acetonitrile or glutathione complex did not become available to E,Zn-SOD1 (Supplementary Fig. 7c). In-cell NMR spectra also showed that in E.coli the SOD1 intrasubunit disulfide bridge is oxidized in a sizable fraction (around 50%, Supplementary Fig. 7d). Unlike bacteria, copper entrance in eukaryotic cells and its delivery to copper-binding proteins require a number of steps, involving specific chaperones responsible of its intracellular trafficking17,18. Accordingly, only around 25% of the recombinant SOD1 protein incorporated copper, again in the Cu(I) state, when human cells were cultured in the presence of Cu(II) (Fig. 2a). The remaining SOD1 fraction contained only one zinc ion per subunit, as observed from the 1H histidine signals in the 1D 1H NMR spectrum (Fig. 2a-c). Additionally, the spectra of the 15N-Cys selectively labelled protein showed only ~20% SOD1 intrasubunit disulfide bond formation (Supplementary Fig. 8). Copper incorporation of SOD1 in eukaryotes is known to be dependent on the CCS protein19,20. Although a basal expression-level of the hCCS gene does occur during normal cell growth, it is likely that the amount of SOD1 produced in our experimental setup was too high to allow for complete copper insertion via the CCS-dependent pathway. However, a CCS-independent copper insertion pathway has also been reported for human SOD121,22 and might have contributed to the partial formation of Cu(I),Zn-SOD1 observed (Fig 2a).

Bottom Line: We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1).We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms.Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

View Article: PubMed Central - PubMed

Affiliation: CERM, Magnetic Resonance Center, University of Florence, Florence, Italy. banci@cerm.unifi.it

ABSTRACT
We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

Show MeSH
Related in: MedlinePlus