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Signals By Kirk Du Plessis
A three-layer hierarchical identification rule based on nine prediction programs was used to identify putative secretory proteins in 158 fungal/oomycete genomes (208,883 proteins, 15.21% of the total proteome). The presence of putative effectors containing known host targeting signals such as RXLX [EDQ] and RXLR was investigated, presenting the degree of bias along with the species. The FSD's user-friendly interface provides summaries of prediction results and diverse web-based analysis functions through Favorite, a personalized repository.
To determine the phylum-level distribution of classes SP, SP3, and SL within fungi, we investigated the proportions of the three classes among subphyla (Figure 2). Class SP3 was the largest, class SP was a little smaller, and the class SL was much smaller; this was consistent over every subphylum. Only in Plasmodium species, oomycetes, and the kingdom Metazoa class SP was dominant. Class SL did not exceeded 2.10% of the whole genome, except in Plasmodium species (4.52%). Plasmodium species also showed the lowest variance among the three classes, which may reflect signal peptide-independent types of secretory proteins such as vacuolar transport signals (VTSs) [12]. These results may be partially affected by the composition of the training data for each prediction program and inherent features of each algorithm.
The SNUGB interface ( )[15] provides several fields: i) signal peptides predicted by four different programs; ii) effector patterns, such as RXLR and RXLX [EDQ]; iii) nucleotide localization signals predicted by predictNLS; iv) transmembrane helixes predicted by TMHMM 2.0c; and v) hydropathy plots (Figure 6). The users can readily compare secretome-related information with diverse genomic contexts.
SNU Genome Browser implemented in the FSD. The SNUGB ( )[15] displays i) four types of signal peptides predicted by SignalP 3.0, SigCleave, SigPred, and RPSP, ii) amino acid patterns, iii) nucleotide localization signals predicted by predictNLS, iv) transmembrane helixes predicted by TMHMM 2.0c, and v) hydropathy plots.
Given the availability of large number of fungal genomes and diverse prediction programs for secretory proteins, a three-layer classification rule was established and implemented in a web-based database, the FSD. With the aid of an automated pipeline, the FSD classifies putative secretory proteins from 158 fungal/oomycetes genomes into four different classes, three of which are defined as the putative secretome. The proportion of fungal secretory proteins and host targeting signals varies considerably by species. It is interesting that fungal genomes have high proportions of the RXLX [EDQ] motif, characterized as host targeting signal in Plasmodium species. Summaries of the complex prediction results from twelve programs help users to readily access to the information provided by the FSD. Favorite, a personalized virtual space in the CFGP, serves thirteen different analysis tools for further in-depth analyses. Moreover, 22 bioinformatics tools provided by the CFGP can be utilized via the Favorite. Given these features, the FSD can serve as an integrated environment for studying secretory proteins in the fungal kingdom.
The desmosomal cadherin Desmoglein-3 (Dsg3) is a core adhesion component in desmosome junctions that occur with high frequency in the stratified squamous epithelial membrane lining the skin and mucous membrane. Dsg3 is identified as a major target of the circulating autoantibodies in Pemphigus Vulgaris (PV), an autoimmune blistering skin disease, and many signaling pathways have been demonstrated to be activated by PV-IgG targeting Dsg3, highlighting its role as a surface regulator in cell signaling. A recent study has revealed an unprecedented role of Dsg3 in the suppression of p53 and shows dysfunction of this pathway in PV. Furthermore, reciprocal crosstalk between p53 and yes-associated protein (YAP) downstream of Dsg3 has been observed in keratinocytes in which increased YAP expression causes suppression of p53 or vice versa. Both p53 and YAP are the crucial nuclear transcription factors involved in regulating cell fate decision, adaptation and tissue integrity in response to environmental and biological cues and are mutually exclusive in human cancer. In this review, we discuss Dsg3 signaling role in keratinocyte response to stress signals, with the highlight on our recent findings of the Dsg3/p53 pathway in the control of cell proliferation and tissue homeostasis, including the DNA integrity, beyond its function in cell-cell adhesion.
The stabilization of p53 is a common response to cellular stress and is one of the key mechanisms by which the p53 function is regulated. Many tumors that retain wild type p53 show defects in this pathway [49]. The stabilization of p53 also seems to be the case in Dsg3 depleted cells since Dsg3 knockdown resulted in an increase of the half-life of p53 protein turnover by approximately two-fold. We showed that such delayed p53 turnover was accompanied by stabilization of MDM2 that may reflect a negative feedback mechanism to control the p53 expression levels [50]. It is worth noting that the stress response of p53 can be obscured by the nature of its fast turnover as we demonstrated by treating cells with the proteasome inhibitor MG132 that revealed greater differences in the levels of p53 expression between the Dsg3 knockdown and control cells [8]. Consistent with the findings from the loss-of-function study, overexpression of Dsg3 resulted in an inverse effect with marked suppression of p53 at both protein and mRNA levels as well as the p53 transcription activity [8]. Furthermore, this regulatory pathway of Dsg3/p53 was consolidated by experiments with several cellular stress responses, such as UV irradiation, genotoxic drug treatment and cyclic mechanical strain, which provoked further enhancement in the levels of p53 and its downstream targets p21Waf1/Cip1 and Bax in cells with Dsg3 knockdown [8]. Collectively, these results suggest strongly that Dsg3 indeed functions as a sensor by modulating the p53 response to stress signals.
The specificity of p53 induction elicited by PV-IgG targeting Dsg3 was verified by additional in vitro studies with PV sera that contain a pool of anti-Dsg3 antibodies (i.e. polyclonal antibodies) and also with the well-characterized specific pathogenic monoclonal antibody AK23 that binds the Dsg3 adhesion site at the N-terminal [74]. The results from both experimental approaches indicated a marked increase of p53 as well as Bax, concomitant with Dsg3 depletion as expected based on several previous studies with PV sera [14,75]. Thus, the observed findings in PV indicate a specific p53 induction associated with PV-IgG induced Dsg3 disturbance as this effect was demonstrated by the treatment of cells with anti-Dsg3 antibody AK23 in a time and dose-dependent manners [8]. Furthermore, it was proved that enhanced p53 is specific since the RNAi mediated p53 knockdown significantly abated the PV sera induced positive p53 signals. These results suggest strongly that activation of the Dsg3/p53 pathway may contribute, at least in part, to PV pathology, with the evidence of early apoptosis that has been shown by others [65,68,76]. This finding may have important implications in clinical diagnosis and also in the development of a novel therapeutic strategy in treating this life-threatening autoimmune disease in the future.
In an attempt to address the involvement of YAP in the Dsg3/p53 pathway, we performed experiments by knocking down of YAP or treating cells with the YAP inhibitor, and our results indicated both approaches caused increased p53 expression and nuclear accumulation (data not shown) (Figure 1). In contrast, transfection of YAP into the Dsg3 depleted cells partially rescued the phenotype of the p53 induction. Furthermore, the antagonistic regulation between YAP and p53 was demonstrated by p53 Luciferase assay that showed inhibition of p53 transcription activity in cells with YAP transfection, with an inverse effect observed in cells with YAP knockdown, as compared to the respective controls. Hence, it is speculated that YAP may bridge in or have an influence on the Dsg3/p53 pathway in keratinocytes, as shown in a working model that Dsg3 restricts p53 via YAP. This simplified model illustrates a relationship among these three signaling molecules in keratinocyte response to stress signals (Figure 1). Notably, this model places Dsg3 upstream of p53 and YAP and indicates that modulation of Dsg3 could have an impact on both signaling pathways, highlighting Dsg3 as an important component of the cellular stress response network in keratinocytes. Hence, this is another example among many other upstream regulators that elaborates reciprocal crosstalk between YAP and p53 for fine-tuning of cell proliferation and apoptosis [81,82].
In summary, our recent studies provide novel evidence that Dsg3 plays a role in regulating p53 response to stress signals in keratinocytes and this pathway likely involves YAP that acts in the suppression of the p53 pathway. Alterations of this pathway may attribute to the pathogenesis of PV where Dsg3 is targeted by autoantibodies resulting in its degradation (loss of function), leading to heightened p53 levels and the activation of the apoptotic machinery. As a consequence, disruption of cell adhesion and cell shrinkage occurs that causes blistering in the Dsg3 baring tissues. The direct evidence of p53 in PV was lacking, and this study fills this gap, suggesting that Dsg3 signaling towards p53 likely reflects cellular stress response in PV. Hence this finding underscores a central role for Dsg3 in pemphigus pathogenesis. The finding also advances our understanding of normal physiological conditions. For instance, the distinct expression patterns of Dsg3 between the skin and mucous membrane may reflect their exposures to different environmental stresses and insults. While the oral mucous membrane is subject to physical and chemical insults daily, the skin is readily prone to UV irradiation with relative less frequency of mechanical stimulation (e.g. trunk skin). Thus this study sheds a light on a potential role for Dsg3 in control of tissue integrity (including the DNA) and homeostasis in these stress-bearing tissues and indicates the pivotal function of Dsg3 as a stress sensor and responder in keratinocytes beyond cell-cell adhesion. 2ff7e9595c
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