SPCA and regucalcin: expression, activity and regulation in Ca2+ homeostasis

Lai, Pei Fong (2011). SPCA and regucalcin: expression, activity and regulation in Ca2+ homeostasis. University of Birmingham. Ph.D.

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Abstract

The secretory pathway Ca\(^{2+}\)-ATPase (SPCA) provides the Golgi apparatus with a luminal Ca\(^{2+}\) store, which is used to modulate the activity of Ca\(^{2+}\)-dependent enzymes involved in controlling the secretory pathway and post-translational modification of proteins. This Ca\(^{2+}\) store controlled by SPCA is also believed to be agonist-releasable. Regucalcin (RGN), (also known as senescence marker protein 30 (SMP30)) is believed to be a Ca\(^{2+}\)-binding protein expressed in an age-dependent manner, whereby its protein levels decrease in a number of organs as aging progresses. It has been suggested to be able to affect the activities of the sarco/endo-plasmic reticulum Ca\(^{2+}\)-ATPase (SERCA), as well as other Ca\(^{2+}\)-dependent enzymes. On the other hand, RGN’s ability to bind Ca\(^{2+}\) has been argued against and this protein has been shown to modulate the activities of enzymes not involved in Ca\(^{2+}\) homeostasis, as well as have intrinsic enzymatic activity in itself as a gluconolactonase. It was of interest in the present studies to examine how SPCA can be regulated and how RGN can contribute to the regulation of Ca\(^{2+}\) homeostasis within intact cells. As a result, the following novel attributes of SPCA and RGN were shown: (1) SPCA expression and activity are sensitive to glucose homeostasis in rat vascular smooth muscle cells (VSMCs); (2) hSPCA1d activity can be inhibited without altering hSERCA2b activity by using bis-phenol and 2-APB; (3) TFP and TBBPA affect both hSPCA1d and hSERCA2b activities to the same degrees, while cADPR and NAADP have no effect on hSPCA1d (and possibly hSERCA2b in the case of cADPR) activity; (4) RGN mRNA expression can be found in some specialised cell types of the male rat reproductive system; and (5) human RGN over-expression can influence Ca\(^{2+}\) mobilisation stimulated by 1\(\mu\)M thapsigargin and 10\(\mu\)M histamine, possibly via alteration of SERCA expression. The present study has also made available a custom-made recombinant form of rat RGN and anti-RGN polyclonal antibodies. It can be strongly suggested that SPCA plays a role in diabetes because of its sensitivity to glucose concentrations, coupled with its involvement in the secretory pathway and ability to influence vasopressin response in VSMCs. Bis-phenol has now been quantitatively shown to be a potent and specific inhibitor of SPCA, giving it great potential to be used as a pharmacological tool for future research on this Ca\(^{2+}\)-ATPase. For RGN, it appears promising that this cytosolic protein has a role to play in male fertility, while its role in Ca\(^{2+}\) homeostasis is likely to involve modulating ER Ca\(^{2+}\) storage capacity and strength of Ca\(^{2+}\)-mediated hormone response.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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