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

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

Gene-delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorous

ABSTRACT Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 ϫ 10 10 infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300-to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300-to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30ϫ LD 50 dose of echothiophate and 5ϫ LD 50 dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5ϫ LD 50 or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same

Purification and properties of synephrinase from Arthrobacter synephrinum

Synephrinase, an enzyme catalyzing the conversion of (−)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (±)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 μmol product formed /min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (−)-synephrine, the enzyme acted upon (±)-octopamine and β-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000

Presence of multiple forms of SMP30 in 7 day old normal rat pup.

<p>Rat liver cytosolic extract in increasing amounts (10, 20 and 30 µg total protein marked as 1, 2 and 3) were subjected to western blotting using antibody against SMP30. Extracts of C3A liver cells expressed SMP30-HA was shown for comparison.</p

Effect of protease inhibitors on the expression and processing of SMP30 in HEK-293A cells.

<p>A, The inhibitors, MGI32 (10 µM), PSI (50 µM), LCys (10 µM), chloroquine (50 µM) and tunicamycin (3 µg/ml) were added along with Ad-SMP30-HA (20 VP/cell) to the cells and incubated for 24 h; B, Effect of proteasomal degradation system on the processing of SMP30 protein expressed in HEK-293A cells in the presence or absence of 10 µM MG132; C, Varying concentrations of calpain inhibitor I (CI, 5, 10, 15 and 20 µM) or MDL28170 (MDL, 5, 10 and 15 µM) were added to the cells along with Ad-SMP30-HA (20 VP/cell) and incubated for 24 h; D, HEK-293A cells were treated with 20 µM caspase inhibitor I [Z-VAD(OMe)-FMK] or 5 µM β-secretase inhibitor (Z-VLL-CHO) or 5 µM γ-secretase inhibitor (Z-LLNle-CHO) or 10 µM MG132 along with Ad-SMP30-HA (20 VP/cell) for 24 h; Equal volume of DMSO served as control. The cells were harvested, lysed with SDS-PAGE buffer containing 5% β-mercaptoethanol and SDS-PAGE followed by western blotting was carried out using anti-HA antibody.</p

Presence of multiple forms of SMP30 in the liver and diaphragm of mice injected with 2×10¹¹ Ad-SMP30-HA per animal.

<p>The animals were injected with the control virus (Ad-null, A & B) or Ad-SMP30-HA (C & D) via the tail vein at a dose of 2×10¹¹ VP/animal and sacrificed on day 4 post-virus injection. Tissue homogenates were processed by western blotting using anti-HA antibody.</p

Schematic diagram showing the gel areas subjected to LC-MS/MS and peptides recovered therefrom.

<p>Schematic diagram showing the gel areas subjected to LC-MS/MS and peptides recovered therefrom.</p

Time course expression of SMP30 and its processed forms in HEK-293A cells and C3A cells.

<p>HEK-293A cell (A) and C3A cells were treated with Ad-SMP30-HA (20 VP/cell for HEK-293A cells and 500 VP/cell for C3A liver cells) for varying periods of time, cell extracts were prepared in M-PER buffer and separated into the particulate and cytosolic fractions. Western blotting was carried out using antibody against HA tag.</p