ABSTRACT
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
Subject(s)
Chromatin/metabolism , Chromosomes, Human/metabolism , Cysteine Endopeptidases/metabolism , Genome, Human , Small Ubiquitin-Related Modifier Proteins/metabolism , Amino Acid Motifs , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Proteins/metabolism , Checkpoint Kinase 1/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cysteine Endopeptidases/chemistry , Genomic Instability , HEK293 Cells , HeLa Cells , Humans , Nuclear Proteins/metabolism , Protein Binding , Sumoylation , Transcription Factors/metabolism , CohesinsABSTRACT
p53 and its cousins p63 and p73 are critical regulators of the genotoxic stress response in mammalian cells. Their activity is controlled by an intricate network of post-translational modifications. The ubiquitin-like SUMO system targets all three family members and modulates their transcriptional activity, stability or subcellular trafficking. While the SUMO system appears to primarily exert a negative regulatory function on p73 and p63, both activating and repressing activities have been reported on p53. Here we will give a synopsis of the multifaceted effects of SUMO on p53 family members.
Subject(s)
Small Ubiquitin-Related Modifier Proteins/physiology , Tumor Suppressor Protein p53/metabolism , Animals , DNA-Binding Proteins/metabolism , Humans , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Protein Stability , Protein Transport , Trans-Activators/metabolism , Transcription Factors , Transcriptional Activation , Tumor Protein p73 , Tumor Suppressor Proteins/metabolismABSTRACT
Attachment of SUMO to proteins regulates protein-protein interactions through noncovalent binding of the SUMO moiety to specialized SUMO interaction motifs (SIMs). A core of hydrophobic amino acids has been described as the major determinant of SIM function. Using the transcriptional coregulator and SUMO ligase PIAS1 as a model, we define an extended phospho-regulated SIM module. We show that serine residues adjacent to the hydrophobic core are phosphorylated by CK2 and demonstrate that this dictates binding of free SUMO and SUMO conjugates to PIAS1 in vivo. We provide evidence that the phosphorylated residues contact lysine 39 and 35 in SUMO1 and SUMO2, respectively. Phospho-dependent SUMO binding does not impair the ligase activity but affects the transcriptional coregulatory potential of PIAS1 and other PIAS family members. CK2-regulated phosphoSIM modules were also dissected in the tumor suppressor PML and the exosome component PMSCL1, indicating that these modules serve as general platforms that integrate CK2- and SUMO-regulated signaling networks.
Subject(s)
Casein Kinase II/metabolism , SUMO-1 Protein/metabolism , Signal Transduction , Binding Sites , Cells, Cultured , HeLa Cells , Humans , Phosphorylation , Protein Inhibitors of Activated STAT/metabolism , Transcription, Genetic , TransfectionABSTRACT
Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4'-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.