The importance of the protein corona formed around nanoparticles upon entering a biological fluid has recently been highlighted. This corona is, when sufficiently long-lived, thought to govern the ...particles’ biological fate. However, even this long-lived “hard” corona evolves and re-equilibrates as particles pass from one biological fluid to another, and may be an important feature for long-term fate. Here we show the evolution of the protein corona as a result of transfer of nanoparticles from one biological fluid (plasma) into another (cytosolic fluid), a simple illustrative model for the uptake of nanoparticles into cells. While no direct comparison can be made to what would happen in, for example, the uptake pathway, the results confirm that significant evolution of the corona occurs in the second biological solution, but that the final corona contains a “fingerprint” of its history. This could be evolved to map the transport pathways utilized by nanoparticles, and eventually to predict nanoparticle fate and behavior.
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IJS, KILJ, NUK, PNG, UL, UM
A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable ...instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
A thorough understanding of how proteins induce nanoparticle （NP） aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also ...crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G （IgG） from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. ...Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Breast cancer is a complex and heterogeneous disease that is usually characterized by histological parameters such as tumor size, cellular arrangements/rearrangments, necrosis, nuclear grade and the ...mitotic index, leading to a set of around twenty subtypes. Together with clinical markers such as hormone receptor status, this classification has considerable prognostic value but there is a large variation in patient response to therapy. Gene expression profiling has provided molecular profiles characteristic of distinct subtypes of breast cancer that reflect the divergent cellular origins and degree of progression.
Here we present a large-scale proteomic and transcriptomic profiling study of 477 sporadic and hereditary breast cancer tumors with matching mRNA expression analysis. Unsupervised hierarchal clustering was performed and selected proteins from large-scale tandem mass spectrometry (MS/MS) analysis were transferred into a highly multiplexed targeted selected reaction monitoring assay to classify tumors using a hierarchal cluster and support vector machine with leave one out cross-validation.
The subgroups formed upon unsupervised clustering agree very well with groups found at transcriptional level; however, the classifiers (genes or their respective protein products) differ almost entirely between the two datasets. In-depth analysis shows clear differences in pathways unique to each type, which may lie behind their different clinical outcomes. Targeted mass spectrometry analysis and supervised clustering correlate very well with subgroups determined by RNA classification and show convincing agreement with clinical parameters.
This work demonstrates the merits of protein expression profiling for breast cancer stratification. These findings have important implications for the use of genomics and expression analysis for the prediction of protein expression, such as receptor status and drug target expression. The highly multiplexed MS assay is easily implemented in standard clinical chemistry practice, allowing rapid and cheap characterization of tumor tissue suitable for directing the choice of treatment.
Blood relations: Nanoparticles that enter the bloodstream become coated with proteins. Four apolipoproteins are consistently recovered on model copolymer nanoparticles by using a centrifugation ...procedure (see electropherogram); their interaction with the nanoparticles is stronger than that of other plasma proteins with higher abundance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
α1-Microglobulin is a 26-kd protein, widespread in plasma and tissues and well-conserved among vertebrates. α1-Microglobulin belongs to the lipocalins, a protein superfamily with highly conserved ...3-dimensional structures, forming an internal ligand binding pocket. The protein, isolated from urine, has a heterogeneous yellow-brown chromophore bound covalently to amino acid side groups around the entrance of the lipocalin pocket. α1-Microglobulin is found in blood both in free form and complex-bound to immunoglobulin A (IgA) via a half-cystine residue at position 34. It is shown here that an α1-microglobulin species, which we name t–α1-microglobulin (t = truncated), with a free Cys34 thiol group, lacking its C-terminal tetrapeptide, LIPR, and with a more polar environment around the entrance of the lipocalin pocket, is released from IgA–α1-microglobulin as well as from free α1-microglobulin when exposed to the cytosolic side of erythrocyte membranes or to purified oxyhemoglobin. The processed t–α1-microglobulin binds heme and the α1-microglobulin–heme complex shows a time-dependent spectral rearrangement, suggestive of degradation of heme concomitantly with formation of a heterogeneous chromophore associated with the protein. The processed t–α1-microglobulin is found in normal and pathologic human urine, indicating that the cleavage process occurs in vivo. The results suggest that α1-microglobulin is involved in extracellular heme catabolism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain ...Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC−MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins. Keywords: calmodulin • calcium • protein−protein interactions • proteomics • brain
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IJS, KILJ, NUK, PNG, UL, UM
Calbindin D 28k (calbindin) is a member of the calmodulin superfamily of Ca 2+ -binding proteins. An intracellular target of calbindin was discovered using bacteriophage display. Human recombinant ...calbindin
was immobilized on magnetic beads and used in affinity purification of phage-displayed peptides from a random 12-mer peptide
library. One sequence, SYSSIAKYPSHS, was strongly selected both in the presence of Mg 2+ and in the presence of Ca 2+ . Homology search against the protein sequence data base identified a closely similar sequence, ISSIKEKYPSHS, at residues
55â66 in myo -inositol-1(or 4)-monophosphatase (IMPase, EC 3.1.3.25 ), which constitute a strongly conserved and exposed region in the three-dimensional structure. IMPase is a key enzyme in
the regulation of the activity of the phosphatidylinositol-signaling pathway. It catalyzes the hydrolysis of myo -inositol-1(or 4)-monophosphate to form free myo -inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems.
Fluorescence spectroscopy showed that isolated calbindin and IMPase interact with an apparent equilibrium dissociation constant,
K
D , of 0.9 μ m . Both apo and Ca 2+ -bound calbindin was found to activate IMPase up to 250-fold, depending on the pH and substrate concentration. The activation
is most pronounced at conditions that otherwise lead to a very low activity of IMPase, i.e. at reduced pH and at low substrate concentration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log ...K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a Ca2+(0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.
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