The Interaction of Piasy with Trim32, an E3-Ubiquitin Ligase Mutated in Limb-girdle Muscular Dystrophy Type 2H, Promotes Piasy Degradation and Regulates UVB-induced Keratinocyte Apoptosis through NF B

Department of Dermatology and Program in Cell and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2006; 281(35):25850-66. DOI: 10.1074/jbc.M601655200
Source: PubMed


Protein inhibitors of activated STATs (PIAS) family members are ubiquitin-protein isopeptide ligase-small ubiquitin-like modifier ligases for diverse transcription factors. However, the regulation of PIAS protein activity in cells is poorly understood. Previously, we reported that expression of Trim32, a RING domain ubiquitin-protein isopeptide ligase-ubiquitin ligase mutated in human limb-girdle muscular dystrophy type 2H (LGMD2H) and Bardet-Biedl syndrome, is elevated during mouse skin carcinogenesis, protecting keratinocytes from apoptosis induced by UVB and tumor necrosis factor-alpha (TNFalpha). Here we report that Trim32 interacts with Piasy and promotes Piasy ubiquitination and degradation. Ubiquitination of Piasy by Trim32 could be reproduced in vitro using purified components. Their interaction was induced by treatment with UVB/TNFalpha and involved redistribution of Piasy from the nucleus to the cytoplasm, where it accumulated in cytoplasmic granules that colocalized with Trim32. Piasy destabilization and ubiquitination required an intact RING domain in Trim32. The LGMD2H-associated missense point mutation prevented Trim32 binding to Piasy, and human Piasy failed to colocalize with human Trim32 in fibroblasts isolated from an LGMD2H patient. Trim32 expression increased the transcriptional activity of NFkappaB in epidermal keratinocytes, both under basal treatment and after UVB/TNFalpha treatment. Conversely, Piasy inhibited NFkappaB activity under the same conditions and sensitized keratinocytes to apoptosis induced by TNFalpha and UVB. Our results indicate that, by controlling Piasy stability, Trim32 regulates UVB-induced keratinocyte apoptosis through induction of NFkappaB and suggests loss of function of Trim32 in LGMD2H.

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    • "There is evidence that a number of the mammalian SUMO E3 ligases are targeted for ubiquitin-dependent proteasomal degradation. For example, PIAS1 is ubiquitinated by human homologs of seven in absentia 2 (hSIAh2) (Depaux et al. 2007), while PIASy is targeted for degradation by the ubiquitin ligase Trim32 (a protein mutated in limb-girdle muscular dystrophy type 2H and in Bardet–Biedl syndrome, and which is also elevated in mouse skin tumours induced in vivo by UVB) (Albor et al. 2006). Treatment of cells with MG132 promoted the interaction of TRIM32 and PIASy, resulting in the colocalisation of both proteins due to the redistribution of PIASy from the nucleus to the cytoplasm. "
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    ABSTRACT: A large number of proteins are modified post-translationally by the ubiquitin-like protein (Ubl) SUMO. This process, known as sumoylation, regulates the function, localisation and activity of target proteins as part of normal cellular metabolism, e.g., during development, and through the cell cycle, as well as in response to a range of stresses. In order to be effective, the sumoylation pathway itself must also be regulated. This review describes how the SUMOylation process is regulated. In particular, regulation of the SUMO conjugation and deconjugation machinery at the level of transcription and by post-translational modifications is discussed.
    Chromosoma 06/2013; 122(6). DOI:10.1007/s00412-013-0422-0 · 4.60 Impact Factor
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    • "Since TRIM32 can ubiquitinate malin substrates, we decided to investigate whether malin could ubiquitinate TRIM32 substrates. It has been described that TRIM32 ubiquitinates, among other substrates, dysbindin (a protein involved in endosomal-lysosomal trafficking and the genetic aetiology of schizophrenia) [33], and PIASy [Protein Inhibitor of Activated STAT (Signal Transducer and Activator of Transcription) isoform y, an E3-SUMO ligase] [34]. Following a similar approach as described above but using myc-dysbindin and myc-PIASy as substrates, we observed that only wild type TRIM32 but not the laforin-malin complex was able to ubiquitinate myc-dysbindin (Figure 8A) and myc-PIASy (Figure 8B). "
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    ABSTRACT: Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin. After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex. We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.
    BMC Evolutionary Biology 07/2011; 11(1):225. DOI:10.1186/1471-2148-11-225 · 3.37 Impact Factor
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    • "The sections were then incubated with affinity-purified chicken anti-Trim32 antibody generated in our laboratory (Albor et al., 2006) or goat anti-CCL20 antibody, clone AF360 (R&D Systems Inc., Minneapolis, MN) overnight at 4°C. The specificity of chicken anti-Trim32 antibody was verified by immunoblotting and immunostaining as described (Albor et al., 2006) and in the Supplemental data herein (Figure S1). The staining signal was visualized by the ABC approach (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. "
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    ABSTRACT: Psoriasis is an inflammatory skin disorder with aberrant regulation of keratinocytes and immunocytes. Although it is well known that uncontrolled keratinocyte proliferation is largely driven by proinflammatory cytokines from the immunocytes, the functional role of keratinocytes in the regulation of immunocytes is poorly understood. Recently, we found that tripartite motif-containing protein 32 (Trim32), an E3-ubiquitin ligase, is elevated in the epidermal lesions of human psoriasis. We previously showed that Trim32 binds to the protein inhibitor of activated STAT-Y (Piasy) and mediates its degradation through ubiquitination. Interestingly, the Piasy gene is localized in the PSORS6 susceptibility locus on chromosome 19p13, and Piasy negatively regulates the activities of several transcription factors, including NF-kappaB, STAT, and SMADs, that are implicated in the pathogenesis of psoriasis. In this study, we show that Trim32 activates, and Piasy inhibits, keratinocyte production of CC chemokine ligand 20 (CCL20), a psoriatic chemokine essential for recruitment of DCs and T helper (Th)17 cells to the skin. Further, Trim32/Piasy regulation of CCL20 is mediated through Piasy interaction with the RelA/p65 subunit of NF-kappaB. As CCL20 is activated by Th17 cytokines, the upregulation of CCL20 production by Trim32 provides a positive feedback loop of CCL20 and Th17 activation in the self-perpetuating cycle of psoriasis.
    Journal of Investigative Dermatology 05/2010; 130(5):1384-90. DOI:10.1038/jid.2009.416 · 7.22 Impact Factor
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