Multitasking in the mitochondrion by the ATP-dependent Lon protease - PubMed (original) (raw)

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Multitasking in the mitochondrion by the ATP-dependent Lon protease

Sundararajan Venkatesh et al. Biochim Biophys Acta. 2012 Jan.

Abstract

The AAA(+) Lon protease is a soluble single-ringed homo-oligomer, which represents the most streamlined operational unit mediating ATP-dependent proteolysis. Despite its simplicity, the architecture of Lon proteases exhibits a species-specific diversity. Homology modeling provides insights into the structural features that distinguish bacterial and human Lon proteases as hexameric complexes from yeast Lon, which is uniquely heptameric. The best-understood functions of mitochondrial Lon are linked to maintaining proteostasis under normal metabolic conditions, and preventing proteotoxicity during environmental and cellular stress. An intriguing property of human Lon is its specific binding to G-quadruplex DNA, and its association with the mitochondrial genome in cultured cells. A fraction of Lon preferentially binds to the control region of mitochondrial DNA where transcription and replication are initiated. Here, we present an overview of the diverse functions of mitochondrial Lon, as well as speculative perspectives on its role in protein and mtDNA quality control.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1. ATP-dependent proteolytic machines within metazoan cells

The 26S proteasome is the only non-mitochondrial energy-dependent protease within animal cells that operates in the cytosol and nucleus. i-AAA and m-AAA are transmembrane proteases located within the mitochondrial inner membrane (IM) with their catalytic sites for ATP hydrolysis and proteolysis located within the intermembrane space (IMS) and matrix, respectively. Lon and ClpXP are soluble proteases within the matrix.

Fig. 2

Fig. 2

(A) Domain structure and functional motifs within the primary amino acid sequence of human mitochondrial Lon. MTS - the mitochondrial targeting sequence of the Lon precursor protein binds to the mitochondrial outer membrane translocation machinery, which mediates protein import into the matrix. N domain- a highly variable amino-terminal domain proposed to function in substrate recognition and binding. AAA+ module- composed of the α/β sub-domain, which contains the Walker Box A and B motifs for ATP -binding and –hydrolysis, in addition to the small α sub-domain. P domain- the protease domain containing the serine (S) and lysine (K) residues forming the catalytic dyad at the proteolytic active sites. S. cerevisiae Lon contains a 54 amino acid charged region that is present between the AAA+ and protease modules. (B) Alignment of the primary amino acid sequences that include the Lon AAA+ module and P-domain from H. sapiens, S. cerevisiae and B. subtilis. Alignment was performed using Jotun-Hein Method. The sequence similarity within this region between H. sapiens and S. cerevisiae and B. subtilis is 58% and 49%, respectively. Walker Box motifs A and B are indicated by a blue line; the highly charged region present in yeast Lon/Pim1p is highlighted in yellow; and the residues forming the serine-lysine dyad are outlined in red.

Fig. 2

Fig. 2

(A) Domain structure and functional motifs within the primary amino acid sequence of human mitochondrial Lon. MTS - the mitochondrial targeting sequence of the Lon precursor protein binds to the mitochondrial outer membrane translocation machinery, which mediates protein import into the matrix. N domain- a highly variable amino-terminal domain proposed to function in substrate recognition and binding. AAA+ module- composed of the α/β sub-domain, which contains the Walker Box A and B motifs for ATP -binding and –hydrolysis, in addition to the small α sub-domain. P domain- the protease domain containing the serine (S) and lysine (K) residues forming the catalytic dyad at the proteolytic active sites. S. cerevisiae Lon contains a 54 amino acid charged region that is present between the AAA+ and protease modules. (B) Alignment of the primary amino acid sequences that include the Lon AAA+ module and P-domain from H. sapiens, S. cerevisiae and B. subtilis. Alignment was performed using Jotun-Hein Method. The sequence similarity within this region between H. sapiens and S. cerevisiae and B. subtilis is 58% and 49%, respectively. Walker Box motifs A and B are indicated by a blue line; the highly charged region present in yeast Lon/Pim1p is highlighted in yellow; and the residues forming the serine-lysine dyad are outlined in red.

Fig. 3

Fig. 3. Molecular modeling of human mitochondrial Lon protease

(A) The ribbon model was generated in PyMol. (B) The space filling model was generated in Schrödinger Suite. Models of the human Lon monomer containing the ATPase and protease domains were initially generated by the CPH server [121], (

http://www.cbs.dtu.dk/services/CPHmodels/

), which uses the SEGMOD tool of GeneMine (Molecular Application Group, GeneMine, Palo Alto, CA, formerly known as XLOOK) [–124]. SEGMOD divides the target sequence into short segments followed by searches for homologous structural fragments in Protein Data Bank (PDB). The fragments are then fitted onto the template structure using secondary structure alignment and multiple independent models were generated of the entire structure. The optimal structure is the average of multiple models (GeneMine Manual, pp. 425 – 426). The resulting structures were submitted to the PDB to identify protein structures with highest homology. A sequence alignment was then generated to identify conserved and defined regions of secondary structure. The secondary structural alignment demonstrated that human Lon has 43.3% identity with Bacillus subtilis Lon (Supplementary Figure 1). By contrast, human Lon showed significantly lower structural identity (36%) with Thermococcus onnurineus NA1 (Ton) Lon. Therefore, the crystal structure of B. subtilis Lon (PDB file 3M6A) [22], was employed as the template structure to model human Lon. The loops joining the secondary structures were modeled by searching for homologous structures in PDB. The side chains were then added using energy constraints. The model obtained from the CPH server was refined using Schrödinger Suite (Schrödinger Inc. LLC, New York, NY). The ‘Prepare Protein’ protocol was used to assign bond orders and valences of each atom, to add the required hydrogen atoms and to optimize H-bond and side chain orientations. The resulting monomer structure was minimized using the ‘Impact’ utility with a OPLS2005 force field. The quaternary structure model of human Lon was generated by Sybyl (Version X; Tripos Associates, St. Louis, MO). A computer program in Sybyl Programming Language (SPL) was written to incorporate a C6 symmetry related operation with energy constraints that limited the steric clash to a distance of 0.5 Å between two side chain atoms at the interface of adjacent monomers in the complex. In the event of steric clashes, energy minimization of the side chains was performed using AMBER force field and Gesteiger-Marshelili charges [125, 126]. The final model was further subject to ‘Protein Preparation’ Wizard’ tool of Schrödinger Suite. A similar protocol was used to generate the model for yeast mitochondrial Lon protease.

Fig. 3

Fig. 3. Molecular modeling of human mitochondrial Lon protease

(A) The ribbon model was generated in PyMol. (B) The space filling model was generated in Schrödinger Suite. Models of the human Lon monomer containing the ATPase and protease domains were initially generated by the CPH server [121], (

http://www.cbs.dtu.dk/services/CPHmodels/

), which uses the SEGMOD tool of GeneMine (Molecular Application Group, GeneMine, Palo Alto, CA, formerly known as XLOOK) [–124]. SEGMOD divides the target sequence into short segments followed by searches for homologous structural fragments in Protein Data Bank (PDB). The fragments are then fitted onto the template structure using secondary structure alignment and multiple independent models were generated of the entire structure. The optimal structure is the average of multiple models (GeneMine Manual, pp. 425 – 426). The resulting structures were submitted to the PDB to identify protein structures with highest homology. A sequence alignment was then generated to identify conserved and defined regions of secondary structure. The secondary structural alignment demonstrated that human Lon has 43.3% identity with Bacillus subtilis Lon (Supplementary Figure 1). By contrast, human Lon showed significantly lower structural identity (36%) with Thermococcus onnurineus NA1 (Ton) Lon. Therefore, the crystal structure of B. subtilis Lon (PDB file 3M6A) [22], was employed as the template structure to model human Lon. The loops joining the secondary structures were modeled by searching for homologous structures in PDB. The side chains were then added using energy constraints. The model obtained from the CPH server was refined using Schrödinger Suite (Schrödinger Inc. LLC, New York, NY). The ‘Prepare Protein’ protocol was used to assign bond orders and valences of each atom, to add the required hydrogen atoms and to optimize H-bond and side chain orientations. The resulting monomer structure was minimized using the ‘Impact’ utility with a OPLS2005 force field. The quaternary structure model of human Lon was generated by Sybyl (Version X; Tripos Associates, St. Louis, MO). A computer program in Sybyl Programming Language (SPL) was written to incorporate a C6 symmetry related operation with energy constraints that limited the steric clash to a distance of 0.5 Å between two side chain atoms at the interface of adjacent monomers in the complex. In the event of steric clashes, energy minimization of the side chains was performed using AMBER force field and Gesteiger-Marshelili charges [125, 126]. The final model was further subject to ‘Protein Preparation’ Wizard’ tool of Schrödinger Suite. A similar protocol was used to generate the model for yeast mitochondrial Lon protease.

Fig. 4

Fig. 4. General paradigm for the recognition and degradation of protein substrates by the Lon protease

Step 1 - recognition and binding of a structural feature or sequence within a protein substrate. **Step 2**- unfolding of structured protein substrates by the AAA+ domains of the holoenzyme, which requires ATP –binding and -hydrolysis. Unfolded substrates or peptides bypass this step. **Step 3**- translocation of unfolded polypeptides or short peptide sequences into the degradation chamber, which also requires ATP –binding and -hydrolysis. Step 4- peptide bond cleavage resulting in the generation of small peptide products. Reiterative rounds of substrate unfolding and translocation are required to degrade substrates completely.

Fig. 5

Fig. 5. Regulation of Lon and ClpXP in the ER and mitochondrial unfolded protein responses

(1) Consequences of the unfolded protein response in the ER (UPRER)(1a) The accumulation of unfolded or misfolded proteins in the ER lumen activates PERK (protein kinase RNA-like endoplasmic reticulum kinase), which phosphorylates and thereby inhibits the translation initiation factor eIF2α. (1b) A block in eIF2α-dependent translation leads to the selective reduction of cytosolic protein synthesis, which includes proteins imported into mitochondria. (1c) The reduction in proteins imported from the cytosol may result in a stoichiometric imbalance of mitochondrial proteins that must assemble to form functional complexes. (1d) Signals originating from the cytosol or the mitochondrion may transcriptionally up-regulate the LONP1 gene by an unknown mechanism. (1e) Alternatively, accumulation of unfolded proteins in the ER directly activates the translation of the transcription factor XBP1 (X-box binding protein 1), which translocates to the nucleus and may activate LONP1 expression by binding to a putative UPRE (unfolded protein response element), which has yet to be identified. (1f) Hypoxia is a physiological inducer of ER stress that has been shown to up-regulate Lon expression by the activation of HIF-1α (hypoxia-inducible factor 1α), which binds to HREs (hypoxia response element) within the promoter of LONP1. (1g) The resulting increase of Lon may function to re-establish mitochondrial homeostasis. (2) Consequences of the unfolded protein response in the mitochondria (UPRMT). (2a) Accumulation of unfolded or misfolded proteins that tax the protein quality control capacity of mitochondria lead to the UPRMT, which is initiated by ClpXP-dependent degradation of protein substrates thereby generating peptides that are effluxed from the matrix to the cytosol by the HAF-1 transporter located in the mitochondrial inner membrane. (2b) The effluxed peptides activate the transcription factors (ZC376.7 and possibly the CHOP/C/EBPβ heterodimer), which then translocate into the nucleus, binding to MURE and CHOP sequences up-regulating the expression of UPRMT related genes encoding ClpP, mtHsp70, mtHsp60, mtHsp10, and mtDnaJ. (2c) The increased expression of ClpP relieves mitochondrial stress and re-establishes mitochondrial homeostasis.

Fig. 6

Fig. 6. Relative locations within the mitochondrial genome of predicted G-quadruplexes and Lon binding

The positions of G-quadruplexes within the heavy-strand of human mtDNA (NC_001807) were predicted using Quadfinder Version 1 (

http://miracle.igib.res.in/quadfinder/

) [16]. The regions of mtDNA bound by Lon in cultured HeLa cells was determined by mtDNA immunoprecipitation (mIP) [15].

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