Kerscher, O. | bioRxiv Science

bioRxiv2021

Chigno/CG11180 and SUMO are Chinmo-Interacting Proteins with a Role in Drosophila Testes Stem Cells

Maintenance of sexual identity on the cellular level ensures the proper function of sexually dimorphic genes expressed in the brain and gonads. Disruption of genes that regulate sex maintenance alters the cellular structure of these tissues and leads to infertility and diseases, such as diabetes, obesity, and gonadal cancers. Sex maintenance in the testis of Drosophila melanogaster depends on the previously identified gene chinmo (Chronologically inappropriate morphogenesis). Chinmos effect on testis differentiation has been investigated in detail, but there is still much to be elucidated about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (Ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmos interaction with CG11180 is SUMOdependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work indicates that SUMO functionally links Chinmo and CG11180 in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Childless Gambino (Chigno) for CG11180.

developmental biology↗

bioRxiv2021

Detection of Rapidly Accumulating Stress-Induced SUMO in Prostate Cancer Cells by a Fluorescent SUMO Biosensor

SUMO conjugates and SUMO chains form when SUMO, a small ubiquitin-like modifier protein, is covalently linked to other cellular proteins or itself. During unperturbed growth, cells maintain balanced levels of SUMO conjugates. In contrast, eukaryotic cells that are exposed to proteotoxic and genotoxic insults mount a cytoprotective SUMO-Stress Response (SSR). One hallmark of the SSR is a rapid and massive increase of SUMO conjugates in response to oxidative, thermal, and osmotic stress. Here, we use a recombinant fluorescent SUMO biosensor, KmUTAG-fl, to investigate differences in the SSR in a normal human prostate epithelial cell line immortalized with SV40 (PNT2) and two human prostate cancer cell lines that differ in aggressiveness and response to androgen (LNCaP and PC3). In cells that grow unperturbed, SUMO is enriched in the nuclei of all three cell lines. However, upon 30 minutes of exposure to ultraviolet radiation (UV) or oxidative stress, we detected significant cytosolic enrichment of SUMO as measured by KmUTAG-fl staining. This rapid enrichment in cytosolic SUMO levels was on average 5-fold higher in the LNCaP and PC3 prostate cancer cell lines compared to normal immortalized PNT2 cells. Additionally, this enhanced enrichment of cytosolic SUMO was reversible as cells recovered from stress exposure. Our study validates the use of the fluorescent KmUTAG-fl SUMO biosensor to detect differences of SUMO levels and localization between normal and cancer cells and provides new evidence that cancer cells may exhibit an enhanced SSR.

cell biology↗