ABSTRACT
Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)- and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration.
Subject(s)
Apoptosis/physiology , Inclusion Bodies/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Mutation, Missense , Nuclear Proteins/metabolism , Oncogene Proteins/genetics , Parkinson Disease , Transcription Factors/metabolism , Antineoplastic Agents/metabolism , Brain Neoplasms , Cell Line , DNA-Binding Proteins , Dopamine/metabolism , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Leupeptins/metabolism , Neuroblastoma , Nuclear Proteins/genetics , Oncogene Proteins/metabolism , Oxidative Stress , Parkinson Disease/genetics , Parkinson Disease/metabolism , Phosphoric Diester Hydrolases , Protein Binding , Protein Deglycase DJ-1 , Substantia Nigra/cytology , Substantia Nigra/metabolism , Transcription Factors/genetics , Two-Hybrid System Techniques , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
GATA-1 is a zinc-finger transcription factor that plays a critical role in the normal development of hematopoietic cell lineages. In human and murine erythroid cells a previously undescribed 40-kDa protein is detected with GATA-1-specific antibodies. We show that the 40-kDa GATA-1 (GATA-1s) is produced by the use of an internal AUG initiation codon in the GATA-1 transcript. The GATA-1 proteins share identical binding activity and form heterodimers in erythroleukemic cells but differ in their transactivation potential and in their expression in developing mouse embryos.
Subject(s)
DNA-Binding Proteins/genetics , Genetic Variation , Peptide Chain Initiation, Translational/genetics , Transcription Factors/genetics , Animals , Base Sequence , Cells, Cultured , DNA-Binding Proteins/metabolism , Embryo, Mammalian/metabolism , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Humans , Mice , Molecular Sequence Data , Molecular Weight , Protein Binding , Protein Conformation , Tissue Distribution , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
The DNA-binding domain of the erythroid transcription factor GATA-1 consists of two closely related, but distinct zinc-fingers which are highly conserved among the members of the growing family of GATA-like factors. The DNA-binding domain of the human GATA-1 (F1F2) was expressed as a histidine-tagged fusion protein in Escherichia coli. The denaturated protein was purified by Ni(2+)-chelate affinity chromatography and renaturated in situ. The active recombinant protein was purified by DNA affinity chromatography. F1F2 displayed GATA-1 specific binding activity toward its DNA recognition sequences within the hypersensitive site 3 of the human locus control region and the human gamma-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F1F2 bound also the CCAAT-box region of the human gamma-globin promoter.
