Effects of peroxynitrite on sarco/endoplasmic reticulum Ca²⁺ pump isoforms SERCA2b and SERCA3a

Abstract

Sarco(endo)plasmic reticulum Ca2+ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca2+ uptake, Ca2+-Mg2+-ATPase, Ca2+-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca2+ uptake, Ca2+-Mg2+-ATPase, and Ca2+-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress.

Platelet Sarcoplasmic Endoplasmic Reticulum Ca 2+ -ATPase and μ-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone

“…These observations are in line with a previous report in native vascular smooth muscle cells in which ONOO Ϫ (10 to 50 mol/L) increased SERCA activity via the S-glutathiolation of critical cysteine residues. 20 Higher concentrations of ONOO Ϫ , on the other hand, enhanced the tyrosine nitration of SERCA-2 in platelets from nondiabetic individuals and decreased platelet Ca 2ϩ -ATPase activity, findings that are consistent with reports in overexpressing systems 21 and in skeletal and smooth muscle. 22,23 At this point, we can only speculate about the in vivo source of the ONOO Ϫ that targets platelets and other plasma proteins and results in their increased tyrosine nitration, 24 but it is certainly possible that it is derived from an uncoupled NOS in either the vascular wall or other circulating cells.…”

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

“…Although SERCA isoforms have a high degree of sequence homology, it appears that distinctive protein modifications occur in different isoforms in response to oxidative stress (12,44); therefore, it was of interest to determine whether Hsp70 could also form a protective interaction with SERCA2a, the SERCA isoform expressed in cardiac muscle. Here we show that Hsp70 can bind directly to SERCA2a and can protect SERCA2a structure and function during heat stress.…”

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

“…The reported higher resistance of SERCA3 versus SERCA2b to oxidative damage might be considered as a meaningful adaptation in these local environments (Grover et al 2003).…”