Molecular Recognition of PTS-1 Cargo Proteins by Pex5p: Implications for Protein Mistargeting in Primary Hyperoxaluria

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.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting

“…Following this view, the peroxisomal import machinery does not seem to show a preference based on the AGT quaternary structure, in line with previous reports [ 63 ]. From a structural-thermodynamic point of view, this agrees with the finding that the principal and ancillary targeting sequences of AGT are not part of the monomer–monomer interface [ 54 , 56 ], thus, indicating that there is no obvious competition between Pex5p binding and AGT oligomerization. Previous studies have focused on the equilibrium of the process, i.e., on the ability of Pex5p to recognize monomeric or oligomerized cargoes, as well as on the role of Pex5p binding in counteracting oligomerization, and contrasting results have been reported.…”

“…AGT folds in the cell cytosol and is then targeted to peroxisomes by a C-terminal PTS1 KKL sequence [ 54 ]. AGT contains one of the weakest PTS1 sequences known for human peroxisomal proteins, as already reported and confirmed by our data ( Table S3 ), but it is efficiently imported due to the high protein levels [ 55 , 56 ]. Consequently, any event that may sequester AGT from the cytosolic pool available for interaction with Pex5 could prevent its proper import.…”

Peroxisome protein import: a complex journey

“…The corresponding PTS1 peptides showed a similar order of binding affinities, but with approximately 10-fold weaker interaction, consistent with the idea that the C-terminus provides the main specificity but that other ancillary regions also contribute to the affinity. They also demonstrated that binding of peptides to PEX5 stabilized the receptor to thermal denaturation and proteolysis with the extent of protection correlating well with the measured binding affinity [ 26 ]. Direct evidence for structural reorganization of the receptor upon binding cargoes has been presented.…”

“…Mis-targeting of AGT to mitochondria due to mutations that increase mitochondrial targeting propensity and destabilize protein structure gives rise to the disorder primary hyperoxaluria type 1. The binding of variant AGT proteins and terminal octapeptides to the TPR domain of PEX5 was compared using isothermal titration calorimetry (ITC), molecular modelling and protein stability assays [ 26 ]. The authors concluded that a consensus PTS1 sequence increased the binding affinity principally by reducing the enthalpic penalty of binding, probably reflecting optimization of binding interactions through changes in conformation and buried surface area.…”

Molecular therapy of primary hyperoxaluria

“…After uptake, the macromolecules are challenged by the acidic pH of endosomes/lysosomes, the digestive enzymes of lysosomes, and the endosomal membrane. For a peroxisomal protein such as AGT, it should be able to escape endosome/lysosome degradation, be stable in the cytosol and interact with the import receptor PEX5P to be eventually targeted at the peroxisome (Mesa-Torres et al 2015). ERT of lysosomal storage diseases has made use of carbohydrate chain modifications in the therapeutic glycoprotein in order to enhance its recognition by endocytic carbohydrate receptors.…”