Most of the methods developed for enzyme extraction
Most of the methods developed for enzyme extraction have focused on the development of aqueous biphasic systems for purification using neutral salts, polymers or ligands (Nadar et al., 2017), similar to those previously described with pineapple and papaya. However, in these cases, only the use of fresh residue for extraction is contemplated, because of the ease with which the enzymes tend to degrade or denature during pretreatment of the same.
Materials and methods
Results and discussion
Introduction Functional plasticity of Imiloxan hydrochloride belonging to the innate and adaptive immune system is necessary for the generation of robust immune responses while minimizing detrimental effects toward the host. CD4+ T cell plasticity has been extensively studied in recent years (O’Shea and Paul, 2010). A plasticity index has been proposed for the various T helper cell lineage subsets, with each subset possessing different lineage flexibility (Murphy and Stockinger, 2010). Of the numerous CD4+ T cell subsets, peripherally generated T regulatory (Treg) cells and T helper (Th) 17 cells are regarded as plastic (Bailey-Bucktrout et al., 2013, Boniface et al., 2010, Gagliani et al., 2015, McGeachy et al., 2007, Mukasa et al., 2010, Yang et al., 2008) whereas functional stability has been attributed toward thymic derived Treg (tTreg) cells (Miyao et al., 2012), Th1, and Th2 cell lineages. Both Th2 cells (Adeeku et al., 2008, Hegazy et al., 2010, Peine et al., 2013, Taylor et al., 2006) and tTreg cells (Feng et al., 2014, Laurence et al., 2012, Zhou et al., 2009) have been demonstrated to be plastic in disease conditions. In light of these studies, Th1 cells remain the lineage with the least evidence of functional flexibility. Furthermore, the molecular mechanisms that influence lineage stability in Th1 cells are poorly defined (Brown et al., 2015). In contrast to work on cytokine signaling (O’Shea and Paul, 2010), the role of co-receptors in mediating functional plasticity has received minimal attention. One such co-inhibitory molecule that has been implicated in Th cell plasticity is programmed death ligand-1 (PDL-1 or B7-H1). In our previous work, we have found that PDL-1 can induce Foxp3 in human Th1 cells (Amarnath et al., 2011), consistent with work in murine naive T cells (Francisco et al., 2009). In the tumor microenvironment, PDL-1 expression coincides with increased intra-tumor Foxp3+ T cells (Duraiswamy et al., 2013, Jacobs et al., 2009), suggesting that PDL-1 may play a role in maintaining Foxp3 expression in CD4+ Th cell subsets. PDL-1 binds to its receptor PD-1 on T cells which signals through the inhibitory phosphatase SHP1 (Chemnitz et al., 2004). SHP1 or SHP2 recruitment results in STAT de-phosphorylation (Amarnath et al., 2011, Taylor et al., 2017), potentially destabilizing the transcriptional signature of Th1 cell lineage. In the current study, we have elucidated an intrinsic mechanism by which PD-1 signaling maintains Foxp3 in Tbet+iTreg and iTreg cells. The data presented here demonstrate that PD-1 can inhibit a functional nuclear pool of active asparaginyl endopeptidase (AEP), an endo-lysosomal protease previously implicated in antigen processing in dendritic cells (Dall and Brandstetter, 2016, Manoury et al., 1998, Manoury et al., 2002). We show that AEP is responsible for destabilizing Foxp3 in both iTreg and Tbet+iTreg cells. We found that PD-1 activation significantly enhanced Foxp3 expression in primed anti-viral and anti-tumor Tbet+Th1 cells, which was reversed in the presence of a blocking antibody to PDL-1. Of note, PDL-1 blockade did not reverse Tbet+Th1 cell conversion and iTreg cell induction in the absence of AEP. Therefore, this study demonstrates that downregulation of AEP is necessary for PD-1-generated Foxp3 stability.