Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/ß-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Lipid Peroxidation Products HNE and ONE Promote and Stabilize Alpha-Synuclein Oligomers by Chemical Modifications.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

Targeted imaging of uPAR expression in vivo with cyclic AE105 variants.

A comprehensive literature reports on the correlation between elevated levels of urokinase-type plasminogen activator receptor (uPAR) and the severity of diseases with chronic inflammation including solid cancers. Molecular imaging is widely used as a non-invasive method to locate disease dissemination via full body scans and to stratify patients for targeted treatment. To date, the only imaging probe targeting uPAR that has reached clinical phase-II testing relies on a high-affinity 9-mer peptide (AE105), and several studies by positron emission tomography (PET) scanning or near-infra red (NIR) fluorescence imaging have validated its utility and specificity in vivo. While our previous studies focused on applying various reporter groups, the current study aims to improve uPAR-targeting properties of AE105. We successfully stabilized the small uPAR-targeting core of AE105 by constraining its conformational landscape by disulfide-mediated cyclization. Importantly, this modification mitigated the penalty on uPAR-affinity typically observed after conjugation to macrocyclic chelators. Cyclization did not impair tumor targeting efficiency of AE105 in vivo as assessed by PET imaging and a trend towards increased tracer uptake was observed. In future studies, we predict that this knowledge will aid development of new fluorescent AE105 derivatives with a view to optical imaging of uPAR to assist precision guided cancer surgery.

EGCG has Dual and Opposing Effects on the N-terminal Region of Self-associating α-synuclein Oligomers.

Oligomers of the protein α-synuclein (α-syn) are thought to be a major toxic species in Parkinson's disease, particularly through their ability to permeabilize cell membranes. The green tea polyphenol epigallocatechin gallate (EGCG) has been found to reduce this ability. We have analyzed α-syn oligomer dynamics and interconversion by H/D exchange monitored by mass spectrometry (HDX-MS). Our results show that the two oligomers OI and OII co-exist in equilibrium; OI is a multimer of OII and its dissociation can be followed by HDX-MS by virtue of the correlated exchange of the N-terminal region. Urea destabilizes the α-syn oligomers, dissociating OI to OII and monomers. Oligomers exposed to EGCG undergo Met oxidation. Intriguingly, EGCG induces an oxidation-dependent effect on the structure of the N-terminal region. For the non-oxidized N-terminal region, EGCG increases the stability of the folded structure as measured by a higher level of protection against H/D exchange. In contrast, protection is clearly abrogated in the Met oxidized N-terminal region. Having a non-oxidized and disordered N-terminal region is known to be essential for efficient membrane binding. Therefore, our results suggest that the combined effect of a structural stabilization of the non-oxidized N-terminal region and the presence of a disordered oxidized N-terminal region renders the oligomers less cytotoxic by decreasing the ability of the N-terminal region to bind to cell membranes and facilitate their permeabilization.

GPIHBP1 and ANGPTL4 Utilize Protein Disorder to Orchestrate Order in Plasma Triglyceride Metabolism and Regulate Compartmentalization of LPL Activity.

Intravascular processing of triglyceride-rich lipoproteins (TRLs) is crucial for delivery of dietary lipids fueling energy metabolism in heart and skeletal muscle and for storage in white adipose tissue. During the last decade, mechanisms underlying focal lipolytic processing of TRLs along the luminal surface of capillaries have been clarified by fresh insights into the functions of lipoprotein lipase (LPL); LPL's dedicated transporter protein, glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1); and its endogenous inhibitors, angiopoietin-like (ANGPTL) proteins 3, 4, and 8. Key discoveries in LPL biology include solving the crystal structure of LPL, showing LPL is catalytically active as a monomer rather than as a homodimer, and that the borderline stability of LPL's hydrolase domain is crucial for the regulation of LPL activity. Another key discovery was understanding how ANGPTL4 regulates LPL activity. The binding of ANGPTL4 to LPL sequences adjacent to the catalytic cavity triggers cooperative and sequential unfolding of LPL's hydrolase domain resulting in irreversible collapse of the catalytic cavity and loss of LPL activity. Recent studies have highlighted the importance of the ANGPTL3-ANGPTL8 complex for endocrine regulation of LPL activity in oxidative organs (e.g., heart, skeletal muscle, brown adipose tissue), but the molecular mechanisms have not been fully defined. New insights have also been gained into LPL-GPIHBP1 interactions and how GPIHBP1 moves LPL to its site of action in the capillary lumen. GPIHBP1 is an atypical member of the LU (Ly6/uPAR) domain protein superfamily, containing an intrinsically disordered and highly acidic N-terminal extension and a disulfide bond-rich three-fingered LU domain. Both the disordered acidic domain and the folded LU domain are crucial for the stability and transport of LPL, and for modulating its susceptibility to ANGPTL4-mediated unfolding. This review focuses on recent advances in the biology and biochemistry of crucial proteins for intravascular lipolysis.