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Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

Mesa-Torres N, Tomic N, Albert A, Salido E, Pey AL - Biomolecules (2015)

Bottom Line: Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation.Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities.Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain. noelmesa@ugr.es.

ABSTRACT
Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

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Thermal stabilization of Pex5p-pbd upon PTS1 nonapeptide binding. (A–E) show thermal denaturation profiles in the absence or presence of different peptide concentrations (as the colors indicated); (F) shows the thermal up-shift (ΔTm) of Pex5p-pbd as a function of peptide concentration.
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biomolecules-05-00121-f008: Thermal stabilization of Pex5p-pbd upon PTS1 nonapeptide binding. (A–E) show thermal denaturation profiles in the absence or presence of different peptide concentrations (as the colors indicated); (F) shows the thermal up-shift (ΔTm) of Pex5p-pbd as a function of peptide concentration.

Mentions: Protein-protein interactions may lead to intracellular stabilization of the protein partners, and alterations in binding and subsequent stabilization may contribute to human disease [24,25]. Stabilization (at least in vitro) is often interpreted as the preferential binding of the partners as folded conformations rather than as non-native conformations, and in principle, the degree of stabilization may be related to binding affinity. We have thus tested whether binding of PTS1 sequences may stabilize Pex5p-pbd using our set of peptides, instead of AGT proteins because the latter show extremely high thermal and kinetic stabilities in vitro [12,26]. To this end, we have used two complementary approaches: thermal denaturation experiments monitored by circular dichroism (Figure 8), and proteolysis kinetics under native conditions (Figure 9).


Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

Mesa-Torres N, Tomic N, Albert A, Salido E, Pey AL - Biomolecules (2015)

Thermal stabilization of Pex5p-pbd upon PTS1 nonapeptide binding. (A–E) show thermal denaturation profiles in the absence or presence of different peptide concentrations (as the colors indicated); (F) shows the thermal up-shift (ΔTm) of Pex5p-pbd as a function of peptide concentration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384115&req=5

biomolecules-05-00121-f008: Thermal stabilization of Pex5p-pbd upon PTS1 nonapeptide binding. (A–E) show thermal denaturation profiles in the absence or presence of different peptide concentrations (as the colors indicated); (F) shows the thermal up-shift (ΔTm) of Pex5p-pbd as a function of peptide concentration.
Mentions: Protein-protein interactions may lead to intracellular stabilization of the protein partners, and alterations in binding and subsequent stabilization may contribute to human disease [24,25]. Stabilization (at least in vitro) is often interpreted as the preferential binding of the partners as folded conformations rather than as non-native conformations, and in principle, the degree of stabilization may be related to binding affinity. We have thus tested whether binding of PTS1 sequences may stabilize Pex5p-pbd using our set of peptides, instead of AGT proteins because the latter show extremely high thermal and kinetic stabilities in vitro [12,26]. To this end, we have used two complementary approaches: thermal denaturation experiments monitored by circular dichroism (Figure 8), and proteolysis kinetics under native conditions (Figure 9).

Bottom Line: Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation.Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities.Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain. noelmesa@ugr.es.

ABSTRACT
Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

Show MeSH
Related in: MedlinePlus