Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA.

ABSTRACT: Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.

SUBMITTER: Chen SH

PROVIDER: S-EPMC2275097 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Co-evolution of multipartite interactions between an extended tmRNA tag and a robust Lon protease in Mycoplasma.

Project description:Messenger RNAs that lack in-frame stop codons promote ribosome stalling and accumulation of aberrant and potentially harmful polypeptides. The SmpB-tmRNA quality control system has evolved to solve problems associated with non-stop mRNAs, by rescuing stalled ribosomes and directing the addition of a peptide tag to the C-termini of the associated proteins, marking them for proteolysis. In Escherichia coli, the ClpXP system is the major contributor to disposal of tmRNA-tagged proteins. We have shown that the AAA+ Lon protease can also degrade tmRNA-tagged proteins, but with much lower efficiency. Here, we present a unique case of enhanced recognition and degradation of an extended Mycoplasma pneumoniae (MP) tmRNA tag by the MP-Lon protease. We demonstrate that MP-Lon can efficiently and selectively degrade MP-tmRNA-tagged proteins. Most significantly, our studies reveal that the larger (27 amino acids long) MP-tmRNA tag contains multiple discrete signalling motifs for efficient recognition and rapid degradation by Lon. We propose that higher-affinity multipartite interactions between MP-Lon and the extended MP-tmRNA tag have co-evolved from pre-existing weaker interactions, as exhibited by Lon in E. coli, to better fulfil the function of MP-Lon as the sole soluble cytoplasmic protease responsible for the degradation of tmRNA-tagged proteins.

| S-EPMC2806816 | biostudies-literature

Characterization of the ATP-Dependent Lon-Like Protease in Methanobrevibacter smithii.

Project description:The Lon protease is highly evolutionarily conserved. However, little is known about Lon in the context of gut microbial communities. A gene encoding a Lon-like protease (Lon-like-Ms) was identified and characterized from Methanobrevibacter smithii, the predominant archaeon in the human gut ecosystem. Phylogenetic and sequence analyses showed that Lon-like-Ms and its homologs are newly identified members of the Lon family. A recombinant form of the enzyme was purified by affinity chromatography, and its catalytic properties were examined. Recombinant Lon-like-Ms exhibited ATPase activity and cleavage activity toward fluorogenic peptides and casein. The peptidase activity of Lon-like-Ms relied strictly on Mg(2+) (or other divalent cations) and ATP. These results highlight a new type of Lon-like protease that differs from its bacterial counterpart.

| S-EPMC4864567 | biostudies-literature

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease.

Project description:Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

| S-EPMC3586414 | biostudies-literature

Vibrio cholerae lon protease mutant microarray analysis

Project description:Lon protease is known to regulate various transcriptional regulators in other bacterial organisms. To understand whether lon protease is involved in transcriptional changes in Vibrio cholerae, wholel-genome level transcriptional profiling was performed using custom microarrays. Transcriptomes of lonA mutant and wild-type strains were compared in this study.

2016-01-06 | GSE73023 | GEO

Vibrio cholerae lon protease mutant microarray analysis

2016-01-06 | E-GEOD-73023 | biostudies-arrayexpress

The Mitochondrial Lon Protease Is Required for Age-Specific and Sex-Specific Adaptation to Oxidative Stress.

Project description:Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. However, a possible molecular mechanism for the sex bias in physiological adaptation to oxidative stress remains unclear. Here, we report that Drosophila melanogaster females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease.

| S-EPMC5224972 | biostudies-literature

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

Project description:We have cloned a human ATP-dependent protease that is highly homologous to members of the bacterial Lon protease family. The cloned gene encodes a protein of 963 amino acids with a calculated molecular mass of 106 kDa, slightly higher than that observed by Western blotting the protein from human tissues and cell lines (100 kDa). A single species of mRNA was found for this Lon protease in all human tissues examined. The protease is encoded in the nucleus, and the amino-terminal portion of the protein sequence contains a potential mitochondrial targeting presequence. Immunofluorescence microscopy suggested a predominantly mitochondrial localization for the Lon protease in cultured human cells. A truncated LON gene, in which translation was initiated at Met118 of the coding sequence, was expressed in Escherichia coli and produced a protease that degraded alpha-casein in vitro in an ATP-dependent manner and had other properties similar to E. coli Lon protease.

| S-EPMC47959 | biostudies-other

The Lon protease links nucleotide metabolism with proteotoxic stress

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) and transposon insertion mutagenesis (Tnseq) libraries of Lon deletions compared to wt Caulobacter crescentus. Methods: See Methods section of The Lon protease links nucleotide metabolism with proteotoxic stress for information regarding methods or contact lead correspondence. Briefly, Samples for RNAseq were extracted from wt and lon deletion strains grown to mid exponential phase. Methods: See Methods section of The Lon protease links nucleotide metabolism with proteotoxic stress for information regarding methods or contact lead correspondence. Briefly, Samples for Tnseq were generated by Eztn5 transposon mutagenesis. Conclusions: Our study represents the first detailed analysis of lon deletion comparison to wt caulobacter transcriptomes, with biologic replicates, generated by RNA-seq technology.

2020-06-14 | GSE152426 | GEO

Mitochondrial cereblon functions as a Lon-type protease.

Project description:Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.

| S-EPMC4945938 | biostudies-literature

Lon protease degrades transfer-messenger RNA-tagged proteins.

Project description:Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

| S-EPMC2045158 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data