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A PROTEIN IN SEARCH OF FUNCTION: NIPSNAP1 IN MITOCHONDRIAL BRANCHED-CHAIN AMINO ACID METABOLON, BRAIN AND APOPTOSIS

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title
A PROTEIN IN SEARCH OF FUNCTION: NIPSNAP1 IN MITOCHONDRIAL BRANCHED-CHAIN AMINO ACID METABOLON, BRAIN AND APOPTOSIS
author
Nautiyal, Manisha
abstract
Four-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) belongs to a highly conserved family of proteins (NIPSNAPs 1-4) of unknown function and shows widespread tissue distribution. We have identified NIPSNAP1 in a protein complex (metabolon) that contains the first two enzymes in branched-chain amino acid (BCAA) catabolism, the mitochondrial branched-chain aminotransferase (BCATm) and the branched-chain α-keto-acid dehydrogenase complex (BCKDC). Dysregulation of BCAA metabolism results in neurologic dysfunction. Studies in brains of phenylketonuria (PKU) and epileptic mice show altered expression of NIPSNAP1. In this study we investigated potential roles for rat NIPSNAP1 in BCAA metabolism and in brain using a variety of different approaches. We characterized the protein-protein interactions of NIPSNAP1 in the BCAA metabolon, investigated NIPSNAP1’s distribution and localization in brain and analyzed its levels in PKU mice. Homologs within the NIPSNAP gene family (NIPSNAP3 & 4) have been reported to function as inhibitors of apoptosis (IAP) antagonists and NIPSNAP1 has also been reported to bind to amyloid precursor protein (APP) which is associated with neurodegeneration in brain. Therefore, we investigated whether NIPSNAP1 could also inhibit IAPs to promote apoptosis focusing on X-linked inhibitor of apoptosis (XIAP), the most potent member of the IAP family. We cloned, overexpressed and purified the recombinant NIPSNAP1 (~30 kDa) and developed antibodies against the recombinant protein. Sedimentation velocity shows that NIPSNAP1 is a homotetramer. It is localized in the mitochondrial matrix where the BCAA enzymes are present and is imported inside mitochondria in an energy-dependent manner. NIPSNAP1 can associate with the E2 (dihydrolipoyl transacylase) component of BCKDC. Overexpression of NIPSNAP1 in human cells results in decreased BCAA oxidation. In rat brain, NIPSNAP1 is expressed exclusively in neurons such as pyramidal neurons in cerebral cortex, Purkinje neurons in cerebellum and motor neurons in spinal cord. Dopaminergic and noradrenergic neurons, which are affected in PKU, also express NIPSNAP1. Brains of PKU mice show gender-specific reduced NIPSNAP1 expression compared to control animals. In human cells, NIPSNAP1 can promote apoptosis by antagonizing XIAP, a key regulator of apoptotic caspase activation. These results suggest a potential role for NIPSNAP1 in BCAA metabolon in situ and apoptosis.
subject
NIPSNAP1
Mitochondria
Neurons
Branched-chain amino acids
Phenylketonuria
Apoptosis
contributor
Leslie B. Poole (committee chair)
Susan M. Hutson (committee member)
R. Mark Payne (committee member)
Raymond Penn (committee member)
Reidar Wallin (committee member)
date
2009-03-30T14:58:29Z (accessioned)
2010-06-18T18:57:47Z (accessioned)
2009-03-30T14:58:29Z (available)
2010-06-18T18:57:47Z (available)
2009-03-30T14:58:29Z (issued)
degree
Molecular Medicine (discipline)
identifier
http://hdl.handle.net/10339/14715 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work for access only to the Wake Forest University system for one year from the date below. After one year, release the entire work for access worldwide. (accessRights)
type
Dissertation

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