Redox control of 20S proteasome gating.
The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities.


OBJECTIVE
The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation.


RESULTS
Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium.


METHODS
We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity.


CONCLUSIONS
S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.
Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, Brasil.
Gustavo M
GM
Silva
Luis E S
LE
Netto
Vanessa
V
Simões
Luiz F A
LF
Santos
Fabio C
FC
Gozzo
Marcos A A
MA
Demasi
Cristiano L P
CL
Oliveira
Renata N
RN
Bicev
Clécio F
CF
Klitzke
Mari C
MC
Sogayar
Marilene
M
Demasi
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D046988:Proteasome Endopeptidase Complex; D005978:Glutathione