A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

244

105

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macromolecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperonelike activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis.

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Rat Liver Peroxisome

Kikuchi

Hatano

Yokota

et al. 2004

244

105

“…Anti-lon protease antibodies were a gift of Michael Maurizi (National Institutes of Health) (28). Anti-nucleolin antibodies were provided by the laboratory of Dr. Nancy Maizels (Yale University) (29).…”

Section: Methodsmentioning

confidence: 99%

IRS Pleckstrin Homology Domains Bind to Acidic Motifs in Proteins

Burks¹,

Wang²,

Towery³

et al. 1998

Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin. Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains. The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in ␤ARK, phospholipase C␥, or spectrin did not bind nucleolin. In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors.Early steps in cellular signaling by growth factors and cytokines are mediated by molecular interactions that are coordinated by common protein modules. In many cases the molecular basis of these protein-protein interactions are known: Src homology 2 domains and phosphotyrosine binding (PTB) 1 domains bind to tyrosine phosphorylated motifs in activated receptors or in transiently associated docking proteins; and Src homology 3 domains and WW domains bind to proline-rich motifs in receptors, enzymes, and the cytoskeleton (1). Pleckstrin homology (PH) domains are also frequent participants in the signaling cascades. Most molecules that contain a PH domain interact with membrane components, suggesting that PH domains mediate membrane targeting. However, PH domains display a broad range of binding specificity, which has hindered the identification of ligands for the various PH domain isoforms.PH domains consist of approximately 100 amino acids, but amino acid sequence alignments reveal few overall identities. A single tryptophan residue in the COOH-terminal ␣-helix is the only residue conserved in all PH domains (2). Secondary structure is the best predictor of the PH domain. X-ray crystallographic and NMR analysis of several PH domains reveal a common structure with two orthogonal ␤-sheets assembled from seven ␤-strands closed at one end with a COOH-terminal ␣-helix (3-8). PH domains contain a positively charged binding pocket, and several reports suggest that these regions bind phospholipids, including the PH domain in PLC␦ that binds to phosphatidylinositol diphosphate and inositol 1,4,5-trisphos...

“…The energy-dependent degradation of casein has been routinely employed to characterize bacterial, yeast, and mammalian Lon (1,8,46,50). The cleavage of FITC-casein by Lon was measured spectrofluorometrically as an increase in acid-soluble fluorescent casein peptides.…”

Section: Purification Of Recombinant Human Lon and Analysis Of Itsmentioning

confidence: 99%

DNA and RNA Binding by the Mitochondrial Lon Protease Is Regulated by Nucleotide and Protein Substrate

Liu

Lü

Lee

et al. 2004

149

134

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GTrich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase ␥ and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.The ATP-dependent Lon (La) protease is a multi-functional enzyme conserved from archaea to mammalian mitochondria (1-6). Mitochondrial Lon is a homo-oligomeric complex in which each monomer carries separate sites for the binding and hydrolysis of both ATP and protein substrate (7,8). Lon selectively degrades abnormal polypeptides, thus serving a quality control function in protein biogenesis (9 -12). The energy requirement of Lon is mechanistically similar to that described for other ATP-dependent proteases such as ClpAP and the proteasome (13)(14)(15)(16)(17)(18)(19)(20). The binding of substrate polypeptide to Lon stimulates its ATPase activity (21). ATP hydrolysis is required for the processive unfolding of a substrate that permits peptide bond cleavage. Conformational changes within the Lon holoenzyme likely coincide with the cycle of ATP binding and hydrolysis, ADP dissociation, and ATP rebinding, as well as with the binding and hydrolysis of protein substrate.Lon belongs to a unique group of proteases that also bind DNA. Other proteases or protease components that bind DNA include the human adenovirus proteinase (AVP), 1 the adipocyte-enhancer binding protein 1 (AEBP1), Gal6p/bleomycin hydrolase, and the 19 S particle of the 26 S proteasome. AVP requires two co-factors for maximal protease activity: viral DNA and a viral peptide produced by AVP proteolysis. It is proposed that AVP utilizes its nonspecific DNA binding activity to locate its viral protein substrates (22). By contrast, the AEBP1 repressor binds specifically to the adipocyte enhancer 1 element. The carboxypeptidase activity of AEBP1 is stimulated by adipocyte enhancer 1 element DNA and is required for transcriptional repression...