ABSTRACT
The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains.
Subject(s)
Adenosine Diphosphate Ribose/chemistry , Poly(ADP-ribose) Polymerases/chemical synthesis , Ubiquitin-Protein Ligases/chemical synthesis , Amino Acid Sequence , Animals , Binding Sites , Computer Simulation , Humans , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Poly(ADP-ribose) Polymerases/chemistry , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Ubiquitin-Protein Ligases/chemistryABSTRACT
Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams-Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys(63) of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination.
Subject(s)
Peptides/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Molecular Sequence Data , Peptides/chemical synthesis , Polyubiquitin/chemistry , Protein Binding , Protein Folding , Protein Structure, Secondary , RING Finger Domains , Substrate Specificity , Transcription Factors/chemical synthesis , Ubiquitin-Conjugating Enzymes/chemical synthesis , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Protein Ligases/chemical synthesis , Ubiquitin-Protein Ligases/chemistry , Ubiquitination , Zinc/chemistryABSTRACT
The creation of the artificial RING finger as ubiquitin-ligating enzyme (E3) has been demonstrated. In this study, by the alpha-helical region substitution between the EL5 RING finger and the Williams-Beuren syndrome transcription factor (WSTF) PHD finger, the artificial E3 (WSTF PHD_RING finger) was newly created. The experiments of the chemical modification of residues Cys and the circular dichroism spectra revealed that the WSTF PHD_RING finger binds two zinc atoms and adopts the zinc-dependent ordered-structure. In the substrate-independent ubiquitination assay, the WSTF PHD_RING finger functions as E3 and was poly- or mono-ubiquitinated. The present strategy is very simple and convenient, and consequently it might be widely applicable to the creation of various artificial E3 RING fingers with the specific ubiquitin-conjugating enzyme (E2)-binding capability.
Subject(s)
RING Finger Domains , Transcription Factors/chemistry , Ubiquitin-Protein Ligases/chemical synthesis , Zinc Fingers , Amino Acid Sequence , Cysteine/chemistry , Humans , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Ubiquitin-Protein Ligases/chemistry , Ubiquitination , Zinc/chemistryABSTRACT
Los sistemas intracelulares proteolíticos reconocen y degradan proteínas lesionadas o mal plegadas. La vía de la ubiquitina proteosoma se encuentra implicada en el recambio intracelular de las proteínas y juega un papel importante en la degradación de proteínas reguladoras de vida corta, implicadas en una serie amplia de procesos celulares tales como: regulación del ciclo celular, modulación de los receptores de superficie y canales iónicos, procesamiento y presentación de antígenos y activacion de factores de transcripción. Esta vía utiliza una cascada enzimática, mediante la cual moléculas de ubiquitina se insertan covalentemente a la proteína sustrato. Un paso importante en la cascada proteolítica es el reconocimiento del sustrato por una de las muchas ubiquitina ligasas, E3, lo cual conduce a la poliubiquitinación o señal de degradación. La modificación por poliubiquitinación marca a la proteína para su destrucción y la conduce al complejo proteosoma 26S para su degradación proteolítica
Intracellular proteolytic systems recognize and destroy misfolded or damaged proteins. The ubiquitin-proteasome pathway is widely involved in intracellular protein turnover. It plays a central role in degradation of short-lived and regulatory proteins important in a broad array of cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, antigen processing and presentation and activation of transcription factors. The pathway employs an enzymatic cascade by which multiple ubiquitin molecules are covalently attached to the protein substrate. An important step in the proteolytic cascade is the specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal.The polyubiquitin modification marks the protein for destruction and directs it to the 26S proteasome complex for proteolytic degradation
