ABSTRACT
Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.
Subject(s)
Proteasome Endopeptidase Complex , Protein Kinases/metabolism , Stress, Physiological/metabolism , Animals , Blotting, Western/methods , Brain/metabolism , Brain/pathology , Brain/ultrastructure , Cell Line , Cloning, Molecular/methods , Cricetinae , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique/methods , Green Fluorescent Proteins/metabolism , Humans , Leupeptins/pharmacology , Microscopy, Immunoelectron/methods , Mitochondria/metabolism , Mitochondria/ultrastructure , Mutant Proteins/genetics , Mutant Proteins/physiology , Nerve Tissue Proteins/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Kinases/genetics , Stress, Physiological/chemically induced , Transfection/methodsABSTRACT
Anp32e/Cpd1, a member of the acidic nuclear phosphoprotein (Anp)32 family, is characterized by the presence of an amino terminal domain containing four leucine-rich repeats and a carboxyl-terminal low-compositional complexity acidic region. In previous studies performed to understand the biological role of Anp32e/Cpd1, we showed a predominant presence of Anp32e/Cpd1 in the nucleus. However, when Anp32e/Cpd1 is in the cytoplasm, it co-localizes spatially with protein phosphatase 2A (PP2A) near cell membranes, far from the synapses. In the present work, we show that Anp32e/Cpd1 is also present as a membrane-bound 74/76-kDa protein with a widespread distribution in the brain. We reveal that the expression, synthesis and half-life of this high-molecular-weight form of Anp32e/Cpd1 are spatially and temporally correlated with the cerebellar synaptogenesis period. We demonstrate that synaptic Anp32e/Cpd1 co-localizes, interacts and inhibits PP2A activity, and that phosphorylation of Anp32/Cpd1 is required for the Anp32e-PP2A interaction. Also, subcellular localization was shown with electronic microscopy. Finally, we examine Anp32e/Cpd1 and PP2A distribution in two ataxic mutant models, weaver and staggerer, and show that their co-localization in Purkinje cell dendrites depends on parallel fibre/Purkinje cell contacts. Based on these observations, we propose that Anp32e/Cpd1 mediates synaptogenesis process by modulating PP2A activity.
Subject(s)
Brain/growth & development , Gene Expression Regulation, Developmental/physiology , Nerve Tissue Proteins/physiology , Phosphoprotein Phosphatases/metabolism , Synapses/metabolism , Age Factors , Animals , Animals, Newborn , Blotting, Western/methods , Brain/cytology , Brain/metabolism , Immunohistochemistry/methods , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Microscopy, Immunoelectron/methods , Molecular Chaperones , Molecular Weight , Organogenesis , Protein Isoforms/metabolism , Protein Phosphatase 2 , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure , Synapses/ultrastructureABSTRACT
Herein we describe the characteristic features of the Anp32 family represented by the cerebellar leucine-rich repeat protein (Lanp) and the cerebellar developmental-regulated protein 1 (Cpd1). The Anp32 family consists of 32 evolutionarily-conserved proteins and is included within the superfamily of leucine-rich repeat (LRR) proteins characterized by the presence of tandem arrays of a LRR, a structural motif implicated in the mediation of protein-protein interactions. We describe three novel human Anp32 proteins, reveal the evolutionary relationships of the members of the Anp32 family, provide insights into their biochemical and structural properties, and review their macromolecular interactions, substrate specificities, tissue distribution/expression patterns, and physiological and pathological roles. Recent findings indicate a conserved role of members of the Anp32 family during evolution in the modulation of cell signalling and transduction of gene expression to regulate the morphology and dynamics of the cytoskeleton, cell adhesion, neural development or cerebellar morphogenesis.
Subject(s)
Cerebellum/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Humans , Leucine-Rich Repeat Proteins , Molecular Chaperones , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/classification , Nuclear Proteins/metabolism , Phylogeny , Protein Structure, Tertiary , Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins.
