Gustavo C. Bressan

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182-GWbs marker protein and TIA-1 stress granule component.

Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution

The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC‐76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N‐terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X‐ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N‐terminus. Ab‐initio 3D low resolution models of the full‐length conformation of the dimeric constructs 6xHis‐FEZ1(1‐392) and 6xHis‐FEZ1(1‐227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C‐terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation. Proteins 2009.

Ki-1/57 interacts with PRMT1 and is a substrate for arginine methylation

The human 57 kDa Ki‐1 antigen (Ki‐1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki‐1/57 interacts with chromo‐helicase DNA‐binding domain proteinu20033 and with the adaptor/signaling protein receptor of activated kinaseu20031 in the nucleus. Among the identified proteins that interacted with Ki‐1/57 in a yeast two‐hybrid system was the protein arginine‐methyltransferase‐1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two‐hybrid system we were able to identify Ki‐1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki‐1/57 and its putative paralog CGI‐55 have two conserved Gly/Arg‐rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine‐methylation by PRMT1. We observed that all Ki‐1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki‐1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine‐2′,3′‐dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki‐1/57, whereas its paralog CGI‐55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two‐hybrid assay is an effective system for identifying novel PRMT arginine‐methylation substrates and may be successfully applied to other members of the growing family of PRMTs.

Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein.

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1, in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki‐1/57 was first identified in malignant cells from Hodgkin’s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki‐1/57 in human cells remains to be determined. Here, we investigated the relationship of Ki‐1/57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki‐1/57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki‐1/57 was able to bind to a poly‐U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki‐1/57 can modify the splicing site selection of the adenoviral E1A minigene in a dose‐dependent manner. Further confocal and fluorescence microscopy analysis revealed the localization of enhanced green fluorescent protein–Ki‐1/57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N‐terminal region. In summary, our findings suggest that Ki‐1/57 is probably involved in cellular events related to RNA functions, such as pre‐mRNA splicing.

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57.

Abstract Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6×His-Ki-1/57(122–413) and 6×His-Ki-1/57(264–413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein β subunit as template. Our model suggests the family-typical seven-bladed β-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

From protein interaction profile to functional assignment: The human protein Ki-1/57 is associated with pre-mRNA splicing events

The mapping of protein-protein interactions of a determined organism is considered fundamental to assign protein function in the post-genomic era. As part of this effort, screenings for pairwise interactions by yeast two-hybrid system have been used popularly to reveal protein interaction networks in different biological systems. Through the identification of protein interaction partners we have successfully obtained interesting functional clues for Ki-1/57, a human protein with no previous functional annotation, in the context of RNA metabolism. We briefly discuss here the way we approached protein-protein interaction data to conduct and interpret further molecular biological and cellular studies as well as structural analyses on this protein. Our data suggest that Ki-1/57 belongs to the family of intrinsically unstructured proteins and that the structural flexibility may be crucial for its capacity to interact with many different proteins. A large fraction of these proteins are involved in pre-mRNA splicing control. Finally, Ki-1/57 is localized to several subnuclear domains, all of which have been described to splicing and other RNA processing events.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkins lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.