Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: an important determinant for their distinctive role in lipid metabolism.

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.

Fluid and condensed ApoA-I/phospholipid monolayers provide insights into ApoA-I membrane insertion.

Apolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoyl-phosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection-absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction.

Surface rheology and adsorption kinetics reveal the relative amphiphilicity, interfacial activity, and stability of human exchangeable apolipoproteins.

Exchangeable apolipoproteins are located in the surface of lipoprotein particles and regulate lipid metabolism through direct protein-protein and protein-lipid interactions. These proteins are characterized by the presence of tandem repeats of amphiphatic alpha-helix segments and a high surface activity in monolayers and lipoprotein surfaces. A noteworthy aspect in the description of the function of exchangeable apolipoproteins is the requirement of a quantitative account of the relation between their physicochemical and structural characteristics and changes in the mesoscopic system parameters such as the maximum surface pressure and relative stability at interfaces. To comply with this demand, we set out to establish the relations among alpha-helix amphiphilicity, surface concentration, and surface rheology of apolipoproteins ApoA-I, ApoA-II, ApoC-I, ApoC-II, and ApoC-III adsorbed at the air-water interface. Our studies render further insights into the interfacial properties of exchangeable apolipoproteins, including the kinetics of their adsorption and the physical properties of the interfacial layer.

Identifying interaction motifs in CK2beta--a ubiquitous kinase regulatory subunit.

Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes: it phosphorylates >300 cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division. CK2 is a heterotetrameric enzyme that induces neoplastic growth when overexpressed. The beta subunit of CK2 (CK2beta) functions as the regulator of the catalytic CK2alpha and CK2alpha' subunits, enhancing their stability, activity and specificity. However, CK2beta also functions as a multisubstrate docking platform for several other binding partners. Here, we discuss the organization and roles of interaction motifs of CK2beta, postulate new protein-interaction sites and map these to the known interaction motifs, and show how the resulting complexity of interactions mediated by CK2 gives rise to the versatile functions of this pleiotropic protein kinase.

Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography.

Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.

MET meet adaptors: functional and structural implications in downstream signalling mediated by the Met receptor.

The tyrosin kinase Met receptor regulates multiple cellular events, ranging from cell motility and angiogenesis to morphological differentiation and tissue regeneration. To conduce these activities, the cytoplasmic C-terminal region of this receptor acts as a docking site for multiple protein substrates, including Grb 2, Gab 1, STAT 3, Shc, SHIP-1 and Src. These substrates are characterised by the presence of multiple domains, including the PH, PTB, SH 2 and SH 3 domains, which directly interact with the multisubstrate C-terminal region of Met. How this receptor recognises and binds a specific substrate in a space-temporal mode is a central question in cell signalling. The recently solved crystal structure of the tyrosine kinase domain of the Met receptor and that of domains of diverse Met substrates provides the molecular framework to understand Met substrate specificity. This structural information also gives new insights on the plasticity of Met signalling and the implications of Met deregulation in tumorigenic processes. In the light of these advances, the present work discusses the molecular basis of Met-substrate recognition and its functional implications in signalling events mediated by this pleiotropic receptor.

On the structure and function of apolipoproteins: more than a family of lipid-binding proteins.

Exchangeable apolipoproteins have been the subject of intense biomedical investigation for decades. However, only in recent years the elucidation of the three-dimensional structure reported for several members of the apolipoprotein family has provided insights into their functions at a molecular level for the first time. Moreover, the role of exchangeable apolipoproteins in several cellular events distinct from lipid metabolism has recently been described. This review summarizes these contributions, which have not only allowed the identification of the apolipoprotein domains that determine substrate binding specificity and/or affinity but also the plausible molecular mechanism(s) involved.