Polyphenolic disaccharides endow proteins with unusual resistance to aggregation

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (<5 mM). This extreme solubilizing activity is a synergistic combination of the glycone and aglycone moieties, as combinations of polyphenols and sugars fail to suppress aggregation. Moreover, the activity of polyphenolic disaccharides is remarkably specific since their monosaccharide counterparts (as well as other common excipients such as arginine, trehalose, and cyclodextrin) fail to prevent aggregation at similar concentrations (<25 mM). We expect that polyphenolic disaccharides will be valuable additives for enhancing the solubility of proteins in applications plagued by protein aggregation.     

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.     

Human insulin receptor substrate-1 (IRS-1) polymorphism G972R causes IRS-1 to associate with the insulin receptor and inhibit receptor autophosphorylation

The most commonly detected polymorphism in human insulin receptor substrate-1 (IRS-1), a glycine to arginine change at codon 972 (G972R), is associated with an increased risk of Type 2 diabetes and insulin resistance. To determine the molecular mechanism by which this polymorphism may be linked to insulin resistance, we produced recombinant peptides comprising amino acid residues 925-1008 from IRS-1 that contain either a glycine or arginine at codon 972 and the two nearby tyrosine phosphorylation consensus sites (EY(941)MLM and DY(989)MTM), which are known binding sites for the p85alpha regulatory subunit of phosphatidylinositol 3-kinase. The wild type peptide could be phosphorylated at these sites in vitro by purified insulin receptor. Introduction of the G972R polymorphism into the peptide reduced the amount of tyrosine phosphorylation by >60%. Pull-down experiments indicated that there was an association between the IRS-1-(925-1008) peptide and the insulin receptor that was markedly enhanced by the presence of the G972R polymorphism. The use of additional overlapping fragments localized this interaction to domains between residues 950-986 of IRS-1 and residues 966-1271 of the insulin receptor, containing the tyrosine kinase domain of the receptor. In addition, the IRS-1-(925-1008) G972R peptide acted as a competitive inhibitor of insulin receptor and insulin-like growth factor-1 receptor autophosphorylation. Taken together, these data indicate that the G972R naturally occurring polymorphism of IRS-1 not only reduces phosphorylation of the substrate but allows IRS-1 to act as an inhibitor of the insulin receptor kinase, producing global insulin resistance.     

Effects of arginine on kinetics of protein aggregation studied by dynamic laser light scattering and tubidimetry techniques

Prevention of undesirable protein aggregation is an extremely important strategy in protein science, medicine, and biotechnology. Arginine is one of the most widely used low molecular weight solution additives effective in suppressing aggregation, assisting refolding of aggregated proteins, and enhancing the solubility of aggregation-prone unfolded molecules in vitro. However, the mechanism of suppression of protein aggregation by arginine is not well understood. To address the mechanism, two model systems have been investigated: protection of alcohol dehydrogenase (ADH) and insulin from heat- and dithiothreitol-induced aggregation, respectively, in the presence of arginine. Using dynamic light scattering (DLS) technique, we have demonstrated the concentration-dependent suppression of light scattering intensity of both ADH and insulin aggregates upon addition of arginine to the incubation medium, a significant effect being revealed in the physiological concentration range of arginine (1-10 mM). DLS studies showed that arginine shifted the populations of nanoparticles with higher hydrodynamic radii to the lower ones, suggesting that the preventive effect of arginine on the protein aggregation process arises because it suppresses intermolecular interactions among aggregation-prone molecules. The results of turbidity measurements were also shown to be consistent with these findings.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with beta isoenzymes and interaction with the catalytic domain of betaII

We analysed the effects of high glucose in rat1 cells overexpressing insulin receptor. High (25 mM) glucose inhibited insulin-stimulated tyrosine kinase activity completely at insulin concentrations of 1 and 5 ng/ml. Decapeptides modelled on insulin receptor sequences surrounding serines 1035 and 1270 were found to inhibit protein kinase C activity in vitro and after microinjection into cells blocked the inhibition of mitogenesis induced by glucose. Purification of receptor from 3T3L1 adipocytes revealed that only the isoenzymes beta1, betaII and delta were detected. The site of the interaction was mapped to the catalytic domain of betaII. These results demonstrate that the inhibition of insulin receptor tyrosine kinase activity can be ameliorated using insulin receptor peptide sequences and there is constitutive and differential interaction of individual PKC isoenzymes with the insulin receptor, and in the case of betaII, this interaction maps to the catalytic domain rather than the regulatory domain.     

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity

We previously reported that novel targeted “hybrid peptide” in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cell lines, and in vivo analysis revealed that this EGFR-lytic peptide displayed significant antitumor activity in a xenograft model of human breast cancer which was resistant to tyrosine kinase inhibitor drugs. As an attempt to improve the selective anticancer activity of EGFR-lytic peptide, we modified the EGFR-binding peptide through introducing the mutation of amino acid according to biophysical analysis by biomolecular interaction and circular dichroism (CD) spectra. When cytotoxic activity of EGFR-lytic or EGFR(2R)-lytic hybrid peptides was investigated in various human cancer and normal cell lines, it was demonstrated that EGFR(2R)-lytic, in which second histidine (H) of EGFR-binding peptide was replaced to arginine (R) had 1.2–1.9-fold higher cytotoxic activity than that of original EGFR-lytic peptide. In vivo analysis also revealed that this modified peptide displayed significant antitumor activity at as low as 1 mg/kg dosage. These results suggest that mutated arginine on EGFR-lytic peptide produces higher binding ability to EGFR on cancer cells, and thereby the improved anticancer activity.     

Peptide mimics of the vesicular stomatitis virus G-protein transmembrane segment drive membrane fusion in vitro.

The efficiency of cell-cell fusion mediated by heterologously expressed vesicular stomatitis virus G-protein has previously been shown to be affected by mutating its transmembrane segment. Here, we show that a synthetic peptide modeled after this transmembrane segment drives liposome-liposome fusion. Addition of millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides suggesting that Ca(2+)-mediated liposome aggregation supports the activity of the peptide. Peptide-driven fusion was suppressed by lysolipid, an established inhibitor of natural membrane fusion, and involved inner and outer leaflets of the liposomal bilayer. Thus, transmembrane segment peptide-driven liposome fusion exhibits important hallmarks characteristic of natural membrane fusion. Importantly, the mutations previously shown to attenuate the function of full-length G-protein in cell-cell fusion also attenuated the fusogenicity of the peptide, albeit in a less pronounced fashion. Therefore, the function of the peptide mimic is dependent on its primary structure, similar to full-length G-protein. Together, our data suggest that the G-protein transmembrane segment is an autonomous functional domain. We propose that it acts at a late step in membrane fusion elicited by vesicular stomatitis virus.     

Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape

Using synthetic peptides, we have identified two distinct regions of the glycoprotein SPARC (Secreted Protein Acidic and Rich in Cysteine) (osteonectin/BM-40) that inhibit cell spreading. One of these sites also contributes to the affinity of SPARC for extracellular matrix components. Peptides representing subregions of SPARC were synthesized and antipeptide antibodies were produced. Immunoglobulin fractions of sera recognizing an NH2-terminal peptide (designated 1.1) blocked SPARC- mediated anti-spreading activity. Furthermore, when peptides were added to newly plated endothelial cells or fibroblasts, peptide 1.1 and a peptide corresponding to the COOH terminal EF-hand domain (designated 4.2) inhibited cell spreading in a dose-dependent manner. These peptides exhibited anti-spreading activity at concentrations from 0.1 to 1 mM. The ability of peptides 1.1 and 4.2 to modulate cell shape was augmented by an inhibitor of protein synthesis and was blocked by specific antipeptide immunoglobulins. In addition to blocking cell spreading, peptide 4.2 competed for binding of [125I]SPARC and exhibited differential affinity for extracellular matrix molecules in solid-phase binding assays. The binding of peptide 4.2 to matrix components was Ca+(+)-dependent and displayed specificities similar to those of native SPARC. These studies demonstrate that both anti- spreading activity and affinity for collagens are functions of unique regions within the SPARC amino acid sequence. The finding that two separate regions of the SPARC protein contribute to its anti-spreading activity lead us to propose that multiple regions of the protein act in concert to regulate the interactions of cells with their extracellular matrix.     

Targeted delivery of a phosphopeptide prodrug inhibits the proliferation of a human glioma cell line

Peptides are ideal candidates for developing therapeutics. Polo-like kinase 1 is an important regulatory protein in the cell cycle and contains a C-terminal polo-box domain, which is the hallmark of this protein family. We developed a peptide inhibitor of polo-like kinase 1 that targets its polo-box domain. This new phosphopeptide, cRGDyK-S-S-CPLHSpT, preferentially penetrates the cancer cell membrane mediated by the integrin receptor, which is expressed at high levels by cancer cells. In the present study, using high performance liquid chromatography and mass spectroscopy, we determined the stability of cRGDyK-S-S-CPLHSpT and its cleavage by glutathione under typical conditions for cell culture. We further assessed the ability of the peptide to inhibit the proliferation of the U87MG glioma cell line. The phosphorylated peptide was stable, and the disulfide bond of cRGDyK-S-S-CPLHSpT was cleaved in 50 mM glutathione. This peptide inhibited the growth of cancer cells and changed their morphology. Therefore, we conclude that the phosphopeptide shows promise as a prodrug and has a high potential to act as an anticancer agent by inhibiting polo-like kinase 1 by binding its polo-box domain. These findings indicate the therapeutic potential of PLHSpT and peptides similarly targeted to surface receptors of cancer cells and to the functional domains of regulatory proteins.     

Regulation of protein arginine methyltransferase 8 (PRMT8) activity by its N-terminal domain

Human protein arginine methyltransferase PRMT8 has been recently described as a type I enzyme in brain that is localized to the plasma membrane by N-terminal myristoylation. The amino acid sequence of human PRMT8 is almost 80% identical to human PRMT1, the major protein arginine methyltransferase activity in mammalian cells. However, the activity of a recombinant PRMT8 GST fusion protein toward methyl-accepting substrates is much lower than that of a GST fusion of PRMT1. We show here that both His-tagged and GST fusion species lacking the initial 60 amino acid residues of PRMT8 have enhanced enzymatic activity, suggesting that the N-terminal domain may regulate PRMT8 activity. This conclusion is supported by limited proteolysis experiments showing an increase in the activity of the digested full-length protein, consistent with the loss of the N-terminal domain. In contrast, the activity of the N-terminal truncated protein was slightly diminished by limited proteolysis. Significantly, we detect automethylation at two sites in the N-terminal domain, as well as binding sites for SH3 domain-containing proteins. We suggest that the N-terminal domain may function as an autoregulator that may be displaced by interaction with one or more physiological inducers.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.     

Mechanism of Free Fatty Acid Effects on Hepatocyte Insulin Receptor Binding and Processing

We determined whether the paimitate effects on hepatocyte insulin receptor binding and post-receptor trafficking were mediated by accelerated mitochondrial (β-oxidation or accumulation of intracellular fatty acyl-CoA derivatives and possibly protein acyiation. Preincubation of hepatocytes with moderate concentrations of paimitate (0.5 mM) resulted in a 23% decline in cell-surface binding and proportional decreases in receptor-mediated insulin internalization and degradation. Brief pretreatment of hepatocytes with the carnitine palmityltransferase-I inhibitor, methyl paimoxirate (MP), prevented 70% of the paimitate effects. At higher paimitate concentrations (2.0 mM), cell-surface binding was reduced by 34%, whereas internalization of the receptor complex was reduced by 78%. These effects were only partially prevented by MP pretreatment. Receptor-mediated insulin degradation increased by 34% and was uninfluenced by MP pretreatment. Octanoate, which is rapidly shunted into mitochondrial oxidation, produced a dose-dependent reduction in insulin binding, with proportional decreases in internalization and degradation. Similarly preincubation with 2.0 mM oleate, which, unlike palmitate, is not known to produce protein acylation, resulted in proportional decreases in insulin receptor binding and receptor-mediated internalization and degradation. High concentrations of octanoate or oleate (2.0 mM) did not reproduce the additive postreceptor effects of palmitate. We conclude that the receptor and post-receptor effects of moderate palmitate concentrations are closely linked to accelerated fatty acid oxidation. The post-receptor effects observed at higher concentrations involve other mechanisms, possibly relating to intracellular levels of palmityl-CoA derivatives.     

Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.     

ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors.     

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.     

Structure of the HIV-1 Full-Length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-Molecule Binding

The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site (“H site”), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by “conformationally trapping” CA in assembly-incompetent conformational states induced by H-site binding.     

Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.     

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.