Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass,surface charge,and nanoparticle–cell interaction

The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood–brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS–PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30 kDa and 100 kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30 kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30 min-incubation.     

Two-hybrid-based analysis of protein–protein interactions of the yeast multidrug resistance protein, Pdr5p

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1) – containing the first NBD domain – and also the C-terminal region of Pdr5p interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein. Electronic Publication     

Engineering cell signaling modulators from native protein–protein interactions

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3′,5′-Cyclic-nucleotide-dependent protein kinase of squamous cell carcinoma of the prostate

An adenosine 3'5'-cyclic-monophosphate (Cyclic AMP)-dependent protein kinase has been identified and partially purified from the rat prostate tumor induced by 20-methylcholanthrene. This enzyme is stimulated 2- to 3-fold by the nucleotide. Equilibrium studies at pH 5.0 suggest the presence of a major class of binding site for cyclic AMP with an association constant of approximately 10(8) M-1. The concentration of binding site is about 1 pmol/mg of protein of the enzyme preparation. The enzyme is stimulated by other cyclic nucleotides as well, but only by higher concentrations. In comparing the ability of different histone subfractions, casein and protamine, to serve as substrate for this particular protein kinase, maximal cyclic-AMP-dependent enzyme activity was observed with histones. The results suggest that factors contributing to the malignant growth of the prostatic tissue do not directly involve changes in the characteristics of a cyclic-AMP-dependent protein kinase.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

Bioluminescence imaging reveals inhibition of tumor cell proliferation by Alzheimer's amyloid β protein

Background  

Cancer and Alzheimer's disease (AD) are two seemingly distinct diseases and rarely occur simultaneously in patients. To explore molecular determinants differentiating pathogenic routes towards AD or cancer, we investigate the role of amyloid β protein (Aβ) on multiple tumor cell lines that are stably expressing luciferase (human glioblastoma U87; human breast adenocarcinoma MDA-MB231; and mouse melanoma B16F).     

Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli

It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Pin1 modulates p63α protein stability in regulation of cell survival,proliferation and tumor formation

The homolog of p53 gene, p63, encodes multiple p63 protein isoforms. TAp63 proteins contain an N-terminal transactivation domain similar to that of p53 and function as tumor suppressors; whereas ΔNp63 isoforms, which lack the intact N-terminal transactivation domain, are associated with human tumorigenesis. Accumulating evidence demonstrating the important roles of p63 in development and cancer development, the regulation of p63 proteins, however, is not fully understood. In this study, we show that peptidyl-prolyl isomerase Pin1 directly binds to and stabilizes TAp63α and ΔNp63α via inhibiting the proteasomal degradation mediated by E3 ligase WWP1. We further show that Pin1 specifically interacts with T₅₃₈P which is adjacent to the P₅₅₀PxY₅₄₃ motif, and disrupts p63α–WWP1 interaction. In addition, while Pin1 enhances TAp63α-mediated apoptosis, it promotes ΔNp63α-induced cell proliferation. Furthermore, knockdown of Pin1 in FaDu cells inhibits tumor formation in nude mice, which is rescued by simultaneous knockdown of WWP1 or ectopic expression of ΔNp63α. Moreover, overexpression of Pin1 correlates with increased expression of ΔNp63α in human oral squamous cell carcinoma samples. Together, these results suggest that Pin1-mediated modulation of ΔNp63α may have a causative role in tumorigenesis.     

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Effect of ionophores on the processing of theβ-amyloid precursor protein in different cell lines

Summary 1. Alzheimer's disease is characterized by the deposition in the brain of extracellular amyloid plaques and vascular deposits consisting mostly of amyloid-peptide (A). A, a polypeptide of 39–43 amino acids (M _r, 4 kDa), is derived proteolytically from a family of proteins of 695–770 amino acids (M _r, 110–140 kDa) called-amyloid precursor protein (APP).2.APP, an integral membrane glycoprotein, is extensively posttranslationally modified within the endoplasmic reticulum (ER) and various Golgi compartments.APP is cleaved by proteases in either the trans-Golgi network or the post-Golgi apparatus and then secreted as a truncated soluble form into the conditioned media of cultured cells and cerebrospinal fluid samples from human subjects.APP can be processed either by an antiamyloidogenic secretory pathway or by an endosomal/lysosomal pathway.3. I studied the effect of two ionophores on the processing ofAPP in cultured cells. Monensin and, in some cases, ammonium chloride increase the intracellular accumulation ofAPP in several cell lines and may alter its processing. Monensin, which had the most consistent effects, also inhibited secretion ofAPP in a differentiated (growth factor mediated) cell line. Nigericin, with greater K⁺ selectivity, was less able to alter the accumulation and possible processing of the protein.4. These results suggest that the increase in the accumulation of intracellularAPP observed after treating cells with ionophores has some specificity. The selective effect of these ionophores on the metabolism ofAPP may provide a model system to analyze the pathways for studying maturation, secretion, and degradation ofAPP.     

The cytoplasmic domain of TGFβR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3^−/− hearts. Tgfbr3^−/− epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3^−/− cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3^−/− cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3^+/+ cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3^−/− mice.     

Maintenance of red blood cell integrity by AMP-activated protein kinase α1 catalytic subunit

AMP-activated protein kinase (AMPK) plays a pivotal role in regulating cellular energy metabolism. We previously showed that AMPKα1−/− mice develop moderate anemia associated with splenomegaly and high reticulocytosis. Here, we report that splenectomy of AMPKα1−/− mice worsened anemia supporting evidence that AMPKα1−/− mice developed a compensatory response through extramedullary erythropoiesis in the spleen. Transplantation of bone marrow from AMPKα1−/− mice into wild-type recipients recapitulated the hematologic phenotype. Further, AMPKα1−/− red blood cells (RBC) showed less deformability in response to shear stress limiting their membrane flexibility. Thus, our results highlight the crucial role of AMPK to preserve RBC integrity.