The closed conformation of the LDL receptor is destabilized by the low Ca<sup>++</sup> concentration but favored by the high Mg<sup>++</sup> concentration in the endosome

Martínez-Oliván, Juan; Arias-Moreno, Xabier; Hurtado‐Guerrero, R.; Carrodeguas, José A.; Miguel-Romero, Laura; Marina, Alberto; Bruscolini, Pierpaolo; Sancho, Javier

doi:10.1016/j.febslet.2015.10.014

Cited by 6 publications

(4 citation statements)

References 49 publications

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“…Upon titration with EDTA, the proteins coexpressed with RAP exhibited disappearance of the band at ∼230 nm and a more significant change of the CD intensity at ∼200 nm than the counterpart proteins, which indicate that the ordered CR domains better retain Ca 2+ ( Table 1 ). These results are consistent with general improvement of binding properties of proteins coexpressed with RAP by SPR in our study, and data of a previous study showed that higher content of Ca 2+ in LDLR favored its binding properties ( 56 ). Thus, coexpression of the proteins with RAP improved their yields and properties.…”

Section: Discussionsupporting

confidence: 93%

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Marakasova

Olivares

Karnaukhova

et al. 2021

Journal of Biological Chemistry

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The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.

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Section: Discussionsupporting

confidence: 93%

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Marakasova

Olivares

Karnaukhova

et al. 2021

Journal of Biological Chemistry

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“…In this state, the LBD folds back onto the β-propeller, forming a loop-like structure with LBD 4 and 5 bound to the β-propeller (Rudenko et al, 2002). This conformational shift allows LDLR to release LDL-C particles for degradation before the receptor is recycled back to the cell membrane (Martínez-Oliván et al, 2015;Tveten et al, 2012). The structural flexibility of the LBD regions largely plays a major role in LDLR adopting an open conformation on the cell membrane and a closed conformation in hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Structural Dynamics of LDL Receptor Interactions with E498A and R499G Variants of PCSK9

Azhar,

Nawawi,

Chua

et al. 2024

Preprint

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The low-density lipoprotein receptor (LDLR) plays an integral role in cellular cholesterol uptake and lipid metabolism by primarily regulating hepatic clearance of plasma low-density lipoprotein cholesterol (LDL-C). Physiologically, proprotein convertase subtilisin/kexin type-9 (PCSK9) attenuates LDLR function by binding to the LDLR extracellular domain, leading to its lysosomal degradation and thereby preventing the total depletion of circulating LDL-C. However, pathogenic variants of PCSK9 are able to reduce the availability of LDLR, thus significantly increasing plasma LDL-C levels. Despite this understanding, the detailed molecular mechanism of LDLR-PCSK9 interaction remains elusive due to the lack of a full atomistic structure of LDLR. In this study, molecular dynamics (MD) simulations were employed to predict LDLR structural dynamics upon binding to PCSK9. Furthermore, two PCSK9 variants, E498A and R499G that were identified in clinically diagnosed Malaysian FH patients were investigated for their mutational effects. The simulations, spanning 500 ns, were conducted for three LDLR-PCSK9 complexes: LDLR-PCSK9 wild-type (WT), LDLR-PCSK9 (E498A), and LDLR-PCSK9 (R499G). Throughout the simulations, PCSK9 structure remained highly stable, in contrast to the LDLR structure that sampled large conformational space. The WT complex exhibited the least change, whereas the R499G complex displayed the most pronounced conformational rearrangement. During the simulations of WT and E498A complexes, the beta-propeller domain of LDLR formed interactions with the prodomain of PCSK9. Aligned with the observation, the MM/GBSA analysis revealed that the E498A complex exhibited the highest LDLR-PCSK9 binding affinity (-63.81 kcal/mol), followed by the WT complex (-33.07 kcal/mol), and the R499G complex (-24.21 kcal/mol). These findings provide novel insights into the dynamic interactions between LDLR and PCSK9, highlighting the importance of structural flexibility in mediating their functional relationship. Further studies with complete LDLR structures are required to fully elucidate the molecular mechanisms underlying LDLR-PCSK9-mediated cholesterol homeostasis.

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“…Both CD320 and LDLR contain LDLR‐A1 and LDLR‐A2 ligand‐binding domains 71,78,79 . However, the selectivity and affinity of CD320 and LDLR are very different: CD320 has the Cbl/TCN2 complex as its only natural ligand, whereas LDLR binds LDL but is not known to bind Cbl/TCN2.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel mechanism for meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP) uptake in cancer cells

Elzi¹,

Bauta²,

Sanchez³

et al. 2021

FASEB j.

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Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin‐mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly (KD=42nM) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.

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The closed conformation of the LDL receptor is destabilized by the low Ca⁺⁺ concentration but favored by the high Mg⁺⁺ concentration in the endosome

Cited by 6 publications

References 49 publications

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Structural Dynamics of LDL Receptor Interactions with E498A and R499G Variants of PCSK9

Identification of a novel mechanism for meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP) uptake in cancer cells

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The closed conformation of the LDL receptor is destabilized by the low Ca++ concentration but favored by the high Mg++ concentration in the endosome

Cited by 6 publications

References 49 publications

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Structural Dynamics of LDL Receptor Interactions with E498A and R499G Variants of PCSK9

Identification of a novel mechanism for meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP) uptake in cancer cells

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The closed conformation of the LDL receptor is destabilized by the low Ca⁺⁺ concentration but favored by the high Mg⁺⁺ concentration in the endosome