Insights into the dynamics of the ATP-dependent human mitochondrial Lon protease by its three-dimensional electron microscopic structural analysis
Project goals
Lon proteases are the most flexible ATP-dependent proteases, which are both in prokaryotic and eukaryotic cells responsible for protein degradation, regulate levels of transcription factors and supposedly participate in apoptosis. Malfunctions of these proteases are linked with dystrophies, neurodegenerative diseases and cancer. A deeper insight into their mechanisms of action requires detailed knowledge of their native three-dimensional (3D) structure in the course of its main functional cycle, the ATP-ase cycle. 3D structure of any flexible Lon protease complex has not been determined so far, however, human mitochondrial Lon protease is the most stable Lon protease purified so far, and therefore we will employ conventional and cryo-electron microscopy in connection with the single particle analysis method to determine the 3D structure of the wild-type of human Lon in the course of its ATP-ase cycle, i.e. in interactions with Mg2+ cations, ATP, ADP and protein substrate, as well as of a few of its available mutant forms, with expected resolutions of ~1nm.
Keywords
Human mitochondrial Lon proteasemitochondria(cryo-) transmission electron microscopysingle particle analysis3D reconstruction
Public support
Provider
Czech Science Foundation
Programme
Post-graduate (doctorate) grants
Call for proposals
Postdoktorandské granty 14 (SGA0201300006)
Main participants
Univerzita Karlova / 1. lékařská fakulta
Contest type
VS - Public tender
Contract ID
13-32339P
Alternative language
Project name in Czech
Pohled do dynamiky ATP-dependentní lidské mitochondriální Lon proteázy trojrozměrnou elektronově mikroskopickou strukturní analýzou
Annotation in Czech
Lon proteázy jsou strukturně nejflexibilnější ATP-závislé proteázy, jež jsou v buňkách prokaryot i eukaryot zodpovědné za degradaci poškozených proteinů, udržují hladinu krátkodobě existujících bílkovin a předpokládá se jejich účast v apoptóze. Poruchy ve funkci těchto proteáz jsou spojovány s dystrofiemi, neurodgenerativními onemocněními a rakovinou. Hlubší porozumění jejich funkčním mechanismům vyžaduje znalost jejich nativní trojrozměrné (3D) struktury v průběhu jejich hlavního funkčního cyklu, tzv. ATP-cyklu. 3D strukturu žádné Lon proteázy se však zatím nepodařilo určit, lidská mitochondriální Lon proteáza je však dosud nejstabilnější izolovanou Lon proteázou, a proto konvenční a kryo-elektronovou mikroskopií ve spojení s metodami single-particle analýzy (SPA) určíme strukturu wild-type Lon v průběhu jejího ATP-cyklu, tj. při interakci s kationty Mg2+, ATP, ADP a proteinovým substrátem, a strukturu několika mutantních forem s předpokládaným rozlišením ~1 nm.
Scientific branches
R&D category
ZV - Basic research
CEP classification - main branch
EB - Genetics and molecular biology
CEP - secondary branch
BO - Biophysics
CEP - another secondary branch
—
10603 - Genetics and heredity (medical genetics to be 3)
10604 - Reproductive biology (medical aspects to be 3)
10605 - Developmental biology
10608 - Biochemistry and molecular biology
10609 - Biochemical research methods
10610 - Biophysics
30101 - Human genetics
Completed project evaluation
Provider evaluation
U - Uspěl podle zadání (s publikovanými či patentovanými výsledky atd.)
Project results evaluation
The results of this three-year postdoctoral project enabled to understand the function of Lon protease, which when expressed more than usual is sensed as a marker of tumour diseases. Received structural information are new and can shift forward the future research on this enzyme. Four publications are dedicated on this project.
Solution timeline
Realization period - beginning
Feb 1, 2013
Realization period - end
Nov 24, 2016
Project status
U - Finished project
Latest support payment
Apr 1, 2016
Data delivery to CEP
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data delivery code
CEP17-GA0-GP-U/01:1
Data delivery date
Jun 30, 2017
Finance
Total approved costs
2,835 thou. CZK
Public financial support
2,835 thou. CZK
Other public sources
0 thou. CZK
Non public and foreign sources
0 thou. CZK
Basic information
Recognised costs
2 835 CZK thou.
Public support
2 835 CZK thou.
100%
Provider
Czech Science Foundation
CEP
EB - Genetics and molecular biology
Solution period
01. 02. 2013 - 24. 11. 2016