A New Function of GAPDH from Chlamydomonas reinhardtii: A Thiol−Disulfide Exchange Reaction with CP12

Abstract

CP12 is a flexible protein that is well-known to interact with GAPDH, and this association is crucial to the regulation of enzyme activity. This regulation is likely related to structural transitions of both proteins, but the molecular bases of these changes are not yet understood. To answer this issue, we undertook a study based on the use of paramagnetic probes grafted on cysteine residues and followed by EPR spectroscopy. We present a new application of this approach that enables us to probe the functional role of cysteine residues in protein-protein interactions. Algal CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state and has some alpha-helical secondary structural elements. In contrast, in its reduced state, CP12 is mainly unstructured and shares some physical properties with intrinsically disordered proteins. Treatment of CP12 with a methane thiosulfonate derivative spin-label (MTSL) led to the labeling of the cysteine residues involved in the C-terminal bridge only as revealed by mass spectrometry. Surprisingly, the partner protein GAPDH induced the cleavage of the disulfide bridge between the cysteine residues of CP12 and the spin-label, resulting in the full release of the label. We showed the existence of a transitory interaction between both proteins and proposed a mechanism based on a thiol-disulfide exchange reaction. The results of this study point out a novel role of the algal GAPDH which is often termed a "moonlighting" protein.

Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: A monolayer study

“…Peptide mass fingerprints were also obtained after tryptic digestion of wtSAL3 and rec(ÀHis)SAL3, using the above mass spectrometer [56]. The proteins digested were studied by performing one-dimensional liquid chromatography (Finnigan Surveyor HPLC Pumps) combined with an ion trap LCQ-DECA XP mass spectrometer (Thermo Finnigan), as previously described [57].…”

“…The proteins digested were studied by performing one-dimensional liquid chromatography (Finnigan Surveyor HPLC Pumps) combined with an ion trap LCQ-DECA XP mass spectrometer (Thermo Finnigan), as previously described [57]. The peptides generated upon trypsin hydrolysis and identified by mass spectrometry were superimposed on the theoretical trypsin cleavage map [56] (Fig. 7).…”

The CP12 protein family: a thioredoxin-mediated metabolic switch?

“…(3) Recent analysis of genome data from 126 species of cyanobacteria has revealed a wide diversity of CP12 protein sequences raising questions about the role of these different CP12 and CP12-like proteins (Stanley et al, 2013). (4) CP12 is a member of a class of proteins known as intrinsically unstructured proteins (IUPs; Gardebien et al, 2006; Erales et al, 2009b; Mileo et al, 2013). This article presents brings together information from a number of recent studies that suggest that CP12 may have a broader role in the regulation of metabolism, over and above the well established role of CP12 in the regulation of the Calvin–Benson cycle.…”

“…Structural and in silico studies have demonstrated that CP12 has physicochemical properties similar to those of IUPs (Graciet et al, 2003; Gardebien et al, 2006; Erales et al, 2009b; Marri et al, 2010; Mileo et al, 2013). IUPs possess relatively little structure in vivo .…”

Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein

“…In the green alga C. reinhardtii, CP12 and GAPDH have complex interactions centered on the Cys pairs. GAPDH can undergo a thiol-disulfide exchange reaction with CP12, cleaving the C-terminal disulfide bond (Erales et al, 2009c). CP12 has also been shown to regulate the Calvin cycle in a redox-independent manner, acting as a specific chaperone for GAPDH to prevent thermal inactivation and aggregation (Erales et al, 2009b).…”