Senescence Marker Protein 30 (SMP30) Expression in Eukaryotic Cells: Existence of Multiple Species and Membrane Localization

Peethambaran Arun,Nageswararao Chilukuri,Vladimir Uversky,Kalpana Parikh,Veeraswamy Manne,Vineela Aleti

doi:10.1371/journal.pone.0016545

Peethambaran Arun, Nageswararao Chilukuri + Show 4 more

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https://doi.org/10.1371/journal.pone.0016545

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Journal: PLoS ONE	Publication Date: Feb 9, 2011
Citations: 74	License type: CC0 1.0

Affiliation: Walter Reed Army Institute of Research

Abstract

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca2+ homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ∼28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of β and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.

Highlights

Senescence marker protein 30 (SMP30) was identified from rat liver in 1992 as an aging factor, the expression of which decreases with age in an androgen independent manner suggesting its possible roles in age related physiologic and pathologic conditions [1,2,3]
The 28 kDa and 24 kDa SMP30 forms were detected in normal rat liver suggesting that native enzyme undergoes a similar processing
Since SMP30 has not been reported to exist in multiple forms and to rule out the possibility that the lower molecular weight forms are an artifact of very high levels of gene expression, we explored for their existence in rat tissues

Summary

Introduction

Senescence marker protein 30 (SMP30) was identified from rat liver in 1992 as an aging factor, the expression of which decreases with age in an androgen independent manner suggesting its possible roles in age related physiologic and pathologic conditions [1,2,3]. Regucalcin was known since 1978 as a calcium-binding protein without the typical Ca2+ binding EF-motif and has been extensively studied for its role in the maintenance of Ca2+ homeostasis and Ca2+ signaling in rat liver and kidney cells [4,5,6,7]. Following the cloning and characterization of genes encoding these proteins, it became clear that SMP30 and regucalcin are one and the same with 299 amino acids and an estimated molecular weight of 33387 Daltons [2,3,4,5,6,7,8]. Immunohistochemical and western blot analysis shows that SMP30 is localized in the cytosol and nucleus of hepatocytes [12] and in the case of kidney, the immunoreactivity was primarily in renal proximal tubular epithelia [2]

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