The maturing oocyte is capable of apoptosis [40]. While MG132 affected relative expression of several proteins involved in apoptosis, it is not clear whether such effects would make the oocyte more or less susceptible to pro-apoptotic signals. MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS [41], HSP90B1 [42], PDIA3 [43], and VCP [44]. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated [45] and one protein increased by MG132, P4HB, is anti-apoptotic [46]. One protein decreased by MG132, VCP, has been implicated as an oocyte-derived sperm attractant in ascidians [47]. It remains to be determined whether this protein plays a similar role in mammals. In any case, addition of MG132 from 16?2 h of maturation did not affect fertilization or alter the rate of polyspermy. The dose-response curve for oocytes exposed to MG132 from 16?2 of maturation was unusual. The optimal beneficial effect was achieved with 10 mM and lower or higher concentrations were not generally effective. Similar effects have been seen in mouse, goat and pig oocytes used for somatic cell nuclear transfer [22,26]Table 7. Proteins whose abundance was altered by 10 mM MG132 from 16?2 h of maturation.
P4HB protein Tubulin alpha-1C chain Cullin-associated NEDD8-dissociated protein 1 Acetyl-CoA acetyltransferase, mitochondrial Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 stress-induced-phosphoprotein 1 ubiquitin-activating enzyme E1 Uncharacterized protein Eukaryotic translation elongation factor 1 gamma (Fragment) bovine serum albumin protein disulfide-isomerase A3 precursor phospholipase A2-activating protein thimet oligopeptidase 1 Cyclin-dependent kinase 5 MFGE8 protein Asparagine synthetase [glutamine-hydrolyzing] Uncharacterized protein MGP57/53 glycoprotein antigen PDIA3 protein Transitional endoplasmic reticulum ATPase Tumor rejection antigen (gp96) 1 Uncharacterized protein as well as for aged mouse oocytes fertilized using intracytoplasmic sperm injection [6]. One possibility is that residual amounts of MG132 in oocytes treated with high concentrations of MG132 interfere with fertilization or subsequent embryonic development. Indeed, functional proteasomes are required for fertilization [48]. One potential use of MG132 is to improve embryo yield from systems of embryo production based on in vitro maturation of
Table 8. Effect of treatment with 10 mM MG132 from 16?2 h of maturation ability on the ability of the resultant blastocysts to establish pregnancy after transfer to recipient females.
oocytes. Results of the embryo transfer experiment reported here indicates that embryos produced from oocytes treated with MG132 from 16?2 h of maturation have the ability to establish pregnancy after transfer to recipients that is generally similar to control embryos. Thus, even though MG132 did rescue some oocytes that might otherwise might not have been fertilized, there was no noticeable decrease in embryo competence for establishment of pregnancy. A larger study with more embryos is needed to verify this observation. In conclusion, our results confirm previous findings that inhibition of proteasomal activity early in oocyte maturation can block progression through meiosis and provide new information that inhibition of proteasomes late in maturation can improve the competence of the oocyte to cleave and the resultant embryo to develop to the blastocyst stage. Such results imply that aging-like effects on the oocyte mediated by proteasomes at the end of maturation can compromise the function of the oocyte and implies that yield of embryos from in vitro embryo production systems can be improved by appropriately-timed treatment with MG132. Results from the embryo transfer experiment would suggest that embryo yield can be increased without a loss of competence to establish pregnancy after transfer to recipients.with iTRAQ tags 114 and 115 and MG132 with iTRAQ tags 116 and 117. A total of 12 fractions were submitted to analysis using a quadrupole TOF MS/MS system. The area coverage of each fraction is shown in panel C. (TIF)
File S1 Results of analysis of the oocyte proteome. The first tab contains data from all proteins detected while the second tab is a subset of data from proteins that were differentially expressed between MG132 and vehicle. Cells in which there was significant increase in relative expression caused by MG132 are highlighted in orange whereas cells in which there was a decrease in relative expression are highlighted in blue. (XLSX) Table S1 Number of proteins identified at critical false discovery rates (FDR) from two databases. (PDF)
Abstract
Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparinactivated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a nonhuman primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFkB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.
Citation: Asmal M, Whitney JB, Luedemann C, Carville A, Steen R, et al. (2012) In vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes. PLoS ONE 7(11): e48234. doi:10.1371/journal.pone.0048234 Editor: Ashok Chauhan, University of South Carolina School of Medicine, United States of America Received January 25, 2012; Accepted September 28, 2012; Published November 2, 2012 Copyright: ?2012 Asmal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the United States National Institutes of Health (N01 AI30048, N01 AI30049, AI067854 and AI060354-06). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
