Background
Ubiquitously eXpressed Transcript isoform 2 (UXT—V2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination.
Methods
We combine co-immunoprecipitation, ubiquitination
in vitro
and
in vivo
, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA.
Results
The Ubl domain of Fbxo7 contributes to interaction with UXT—V2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages
in vitro
and
in vivo
. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXT—V1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXT—V2, also interacts with and is ubiquitinated by Fbxo7. Moreover,
FBXO7
knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus.
Conclusions
Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway.
General significance
Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.
Partial characterization of SCF1 complex containing the protein phosphorylated FBXO25. Medeiros, A. C., 2015. Dissertação de mestrado 73p. Programa de Pós-graduação em Bioquímica da FMRP/USP. The FBXO25 is an E3 ligase RING type (Really Interesting New Gene), SCF oligomeric type, responsible for the specific recognition of the substrate to be degraded via the ubiquitinproteasome system (SUP). SUP is the main intracellular proteolytic mechanism responsible for 80-90% degradation of cytosolic and nuclear proteins. The FBXO25 is capable of forming a complex SCF1 (formed by the interaction of proteins Skp1, Cul1, Roc1 protein and a type Fbox), resulting in an active SCF complex which is able to ubiquitinate their substrates. This protein accumulates in the nucleus forming a subnuclear structure called FANDs (FBXO25 Associated Nuclear Domains) that are involved in nuclear ubiquitination. In this work, we purify complex SCF1 (WT or ΔF-box, which is not able to interact with Skp1), treated or not with PMA, by immunoprecipitation technique. We identified by mass spectrometry, an essential phosphorylation site for FBXO25, when it is phosphorylated under the action of the mitogenic reagent PMA. We also search for differentially ubiquitinated substrates for these complexes, by testing in ProtoArrays® identifying substrates involved in the signaling pathway ERK1 / 2.
Biochemical and subcellular characterization of SAMHD1 protein Medeiros, A. C., 2019. Tese de doutorado 93p. Programa de Pós-graduação em Bioquímica da FMRP/USP. SAMHD1 is a 626 amino acid protein in an N-terminal SAM motif and a domain containing histidine and aspartic acid (HD). The SAM domain is involved in protein-protein and protein-DNA / RNA interactions. Proteins with the HD domain are part of a superfamily of phosphohydrolases commonly involved in the metabolism of nucleic acids. SAMHD1 has the function of decreasing the pool of intracellular dNTPs, and mutations in the SAMHD1 gene are associated with Aicardi-Goutières Syndrome (AGS). In this work, we evaluated the specificity of commercial anti-SAMHD1 antibodies in different cell lines, determined their subcellular location in response to DNA damage induced by camptothecin (CPT) that induces double strand breaks (DSBs). Finally, we investigated the possibility of an endogenous truncated alternative form of SAMHD1. We have seen that the anti-SAMHD1 monoclonal antibody has cross-reactivity with a protein of approximately 50 kDa, of yet unknown function, whereas the polyclonal antibody specifically recognizes a band of molecular mass corresponding to SAMHD1. In addition, we have identified that the overexpression of SAMHD1 produces a truncated form SAMHD1 (ATG2) without the first 114 amino acids, including the SAM domain. This form was produced thanks to an alternative start codon located at 345 nucleotides upstream of the ATG1, SAMHD1 gene. Finally, we conclude that SAMHD1 activates the response to the damage, but it is not recruited to the nuclear foci, different from what has recently been published in the literature.
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