11 research outputs found
The Absence of Indoleamine 2,3-Dioxygenase Inhibits Retinal Capillary Degeneration in Diabetic Mice.
Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function
Abstractα-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein
Recommended from our members
The Absence of Indoleamine 2,3-Dioxygenase Inhibits Retinal Capillary Degeneration in Diabetic Mice
Purpose:Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods:IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results:The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions:The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR
The Absence of Indoleamine 2,3-Dioxygenase Inhibits Retinal Capillary Degeneration in Diabetic Mice
Purpose:Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods:IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results:The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions:The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR
Acetylation of Lysine 92 Improves the Chaperone and Anti-apoptotic Activities of Human αB-Crystallin
αB-Crystallin
is a chaperone and an anti-apoptotic protein that is strongly expressed
in many tissues, including the
lens, retina, heart, and kidney. In the human lens, several lysine
residues in αB-crystallin are acetylated. We have previously
shown that such acetylation is
predominant at lysine 92 (K92) and lysine 166 (K166). We have investigated
the effect of lysine acetylation on the structure and functions of
αB-crystallin
by the specific introduction of an <i>N</i><sup>ε</sup>-acetyllysine (AcK) mimic at K92. The introduction of AcK slightly
altered the secondary and tertiary structures of the protein. The
introduction of AcK also resulted in an increase in the molar mass
and hydrodynamic radius of the protein, and the protein became structurally
more open and more stable than the native protein. The acetyl protein
acquired higher surface hydrophobicity and exhibited 25–55%
higher chaperone activity than the native protein. The acetyl protein
had more client protein binding per subunit of the protein and higher
binding affinity relative to that of the native protein. The acetyl
protein was at least 20% more effective in inhibiting chemically induced
apoptosis than the native protein. Molecular modeling suggests that
acetylation of K92 makes the “α-crystallin domain”
more hydrophobic. Together, our results reveal that
the acetylation of a single lysine residue in αB-crystallin
makes the protein structurally more
stable and improves its chaperone and anti-apoptotic activities. Our
findings suggest that lysine acetylation of αB-crystallin is
an important chemical modification for enhancing αB-crystallin’s
protective functions in the eye