Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western wo... more Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome in vitro. Animals were treated with ethanol (3.5 g/kg) or saline between postnatal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids. Twenty-four hour conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here, we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among the 59 proteins that were found to be reduced in ET-CM we observed components of the extracellular matrix such as laminin subunits α2, α4, β1, β2, and γ1 and the proteoglycans biglycan, heparan sulfate proteoglycan 2, and lumican. Proteins with trophic function such as insulin-like growth factor binding protein 4, pigment epithelium-derived factor, and clusterin as well as proteins involved on modulation of proteolysis such as metalloproteinase inhibitor 1 and plasminogen activator inhibitor-1 were also reduced. In contrast, pro-synaptogeneic proteins like thrombospondin-1, hevin as well as the modulator of extracelular matrix expression, angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through ingenuity pathway analysis demonstrated that the amyloid beta A4 protein precursor, which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM. Taken together our results strongly suggest that early exposure to teratogens such as alcohol may lead to an enduring change in astrocyte secretome. Despite efforts in prevention, fetal alcohol spectrum disorders are a major cause of mental disabilities. Here, we show that developmental exposure to alcohol lead to a persistent change in the pattern of proteins secreted (secretome) by astrocytes. This study is also the first mass spectrometry-based assessment of the astrocyte secretome in a gyrencephalic animal. Cover Image for this issue: doi: 10.1111/jnc.13320.
Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a nu... more Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.
Biochemical and Biophysical Research Communications, Dec 15, 1988
We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors ... more We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors (IGFs) from BRL-3A cell conditioned medium. The fragment binds IGF-I and IGF-II with similar specificity to the 30 kDa binding protein, but with lower affinity. It corresponds to the carboxy terminus of the native binding protein (residues 148-270), and is thought to arise by proteolysis. We infer that this region of the native binding protein contains, at least in part, the IGF binding domain, o J.988 Academic P ..... Tnc.
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were as...
The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for pr... more The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% ...
Insulin-like growth factor II is a growth factor important in fetal development. Several cancer t... more Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observati...
Integrin receptors mediate the formation of adhesion complexes and play important roles in signal... more Integrin receptors mediate the formation of adhesion complexes and play important roles in signal transduction from the extracellular matrix. Integrin-based adhesion complexes (IAC) contain proteins that link integrins to the cytoskeleton and recruit signaling molecules, including vinculin, paxillin, focal adhesion kinase, talin and alpha-actinin. In this study, we describe a approximately 160 kDa protein that is markedly enriched at IAC induced by clustering integrins with fibronectin-coated beads. Protein sequence analysis reveals that this approximately 160 kDa protein is kinectin. Kinectin is an integral membrane protein found in endoplasmic reticulum, and it serves as a receptor for the motor protein kinesin. Fibronectin-induced IAC sequestered over half of the total cellular content of kinectin within 20 minutes. In addition, two other ER-resident proteins, RAP [low-density lipoprotein receptor-related protein (LRP) receptor-associated protein] and calreticulin, were found to ...
Current protocols in protein science / editorial board, John E. Coligan ... [et al.], 2011
Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are prese... more Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.
Proceedings of the National Academy of Sciences, 2006
RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of... more RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene-and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to
Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affe... more Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the a catalytic subunit of protein kinase CK2 (CK2a) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2a and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.
Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplast... more Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institutions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth muscle cell (SMC) proliferation, but do not effect endothelial cell (EC) proliferation. The purposes of this study were to (1) quantify the diffusive release of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) determine the effect of heparin and FGF-1 on SMC proliferation when the cells are immediately plated on the FG; and (3) by means of the diffusive release data, design a new in vitro model that may differentiate the effect of FG-incorporated FGF-1 and heparin, rather than the released, solubilized components of these two factors, on SMC and EC proliferation. 125I-FGF-1 or 3H-heparin release from FG into the overlying media was measured serially in a 96-hour period, either with or without cells. SMCs were immediately plated on FG containing various concentrations of FGF-1 and heparin. SMCs or ECs were plated on identical groups of FG containing FGF-1 and heparin 24 hours after the FG was made to exclude the effect on cell growth of the initial release of FGF-1 into the media. In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of the heparin in the FG was released into the overlying media, with minimal release occurring thereafter. The cell type or absence of cells did not affect release, but there was five times more FGF-1 and four times more heparin in the media at 72 hours for the immediate plating versus the delayed plating because of a diffusive release primarily in the first 24 hours. A heparin concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing immediate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, immediate plating induced greater proliferation than delayed plating; this was likely caused by the higher soluble FGF-1 concentration. Heparin doses as high as 500 U/mL had little effect on SMC proliferation. In contrast, ECs died with delayed plating on FG containing 500 U/mL heparin, and their growth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin. The differences in SMC proliferation when comparing immediate versus delayed plating are likely caused by diffusive release of heparin and FGF-1 into the media. Our ongoing work uses an optimized in vitro FG system that minimizes the effects of soluble factors. This is an important distinction, because the cytokines that are released in vivo will be removed by blood flow and, thus, may not exert an effect unless they are contained within the FG.
Background. The structure/function relationships of fibroblast growth factor 1 (FGF-1) are being ... more Background. The structure/function relationships of fibroblast growth factor 1 (FGF-1) are being investigated using site mutation, yielding novel structures with potential clinical applicability for modulating tissue responses to vascular interventions. We generated a mutant FGF-1 in which all three cysteines were converted to serines and then tested the relative mitogenic activities on endothelial cells (ECs) and smooth muscle cells (SMCs) and the molecular stability of the protein to thrombin-induced degradation.
The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angi... more The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-type HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity. Thus, the role of nuclear translocation in the mechanisms of action of HBGF-1 remains unclear.
The contribution of individual basic amino acids within three putative "consensus sequences" for ... more The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.
We have previously characterized the release of the signal peptide sequence-less fibroblast growt... more We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation.
In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (a... more In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (appMr) of 15,000 precursor form of human insulin-like growth factor-II (hIGF-II) was shown to be greater than that of completely processed hIGF-II for human fetal-derived fibroblasts, and both were more potent than rIGF-I. Since it is generally acknowledged that the stimulation of cell replication by the IGFs is mediated by IGF-I receptors, we undertook to determine whether differences between the receptors' affinity for the two Mr forms of hIGF-II and recombinant IGF-I (rIGF-I) or between its efficiency to couple specific growth factor occupancy to the activation of protein kinase could explain the greater replicating potential of appMr 15,000 hIGF-II. Equilibrium dissociation, i.e. Kd, and inhibition, i.e. Ki, constants were determined by measuring the ability of rIGF-I, hIGF-II, appMr 15,000 hIGF-II, insulin, and the antireceptor monoclonal antibody alpha IR-3 to compete with 125I-labeled rIGF-I and hIGF-II for binding to purified preparations of IGF-I receptors prepared from an enriched source of fetal membrane, i.e. human term placenta. The results of these experiments established that 1) hIGF-II and appMr 15,000 hIGF-II bind to the IGF-I receptor with the same affinity as rIGF-I, e.g. with Kd and Ki values between 0.03-0.07 nM; 2) the total binding capacity, i.e. Ro, for IGF-I binding was not statistically different from the Ro calculated for IGF-II binding; and 3) the statistical analysis of 12 data sets from the competitive binding experiments for goodness of fit indicated that a 1-site model for IGF-I and -II binding was a better fit of the data than a 2-site model. Measurements of the stimulation of IGF-I receptor autophosphorylation at low ligand concentrations established that appMr 15,000 hIGF-II and hIGF-II were more effective than rIGF-I in coupling receptor occupancy to the activation of its protein kinase. At saturating ligand concentrations, the 3 had similar potencies. The original preparation of appMr 15,000 hIGF-II contains a mixture of forms with acidic isoelectric points (pIs) and was more potent than Mr 7,500 IGF-II in stimulating receptor autophosphorylation. These results are consistent with the relative potencies of this preparation, hIGF-II, and rIGF-I in stimulating the replication of 12-week-old fetal dermal fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)
A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to hom... more A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.
Biochemical and Biophysical Research Communications, 1988
We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors ... more We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors (IGFs) from BRL-3A cell conditioned medium. The fragment binds IGF-I and IGF-II with similar specificity to the 30 kDa binding protein, but with lower affinity. It corresponds to the carboxy terminus of the native binding protein (residues 148-270), and is thought to arise by proteolysis. We infer that this region of the native binding protein contains, at least in part, the IGF binding domain, o J.988 Academic P ..... Tnc.
Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western wo... more Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome in vitro. Animals were treated with ethanol (3.5 g/kg) or saline between postnatal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids. Twenty-four hour conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here, we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among the 59 proteins that were found to be reduced in ET-CM we observed components of the extracellular matrix such as laminin subunits α2, α4, β1, β2, and γ1 and the proteoglycans biglycan, heparan sulfate proteoglycan 2, and lumican. Proteins with trophic function such as insulin-like growth factor binding protein 4, pigment epithelium-derived factor, and clusterin as well as proteins involved on modulation of proteolysis such as metalloproteinase inhibitor 1 and plasminogen activator inhibitor-1 were also reduced. In contrast, pro-synaptogeneic proteins like thrombospondin-1, hevin as well as the modulator of extracelular matrix expression, angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through ingenuity pathway analysis demonstrated that the amyloid beta A4 protein precursor, which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM. Taken together our results strongly suggest that early exposure to teratogens such as alcohol may lead to an enduring change in astrocyte secretome. Despite efforts in prevention, fetal alcohol spectrum disorders are a major cause of mental disabilities. Here, we show that developmental exposure to alcohol lead to a persistent change in the pattern of proteins secreted (secretome) by astrocytes. This study is also the first mass spectrometry-based assessment of the astrocyte secretome in a gyrencephalic animal. Cover Image for this issue: doi: 10.1111/jnc.13320.
Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a nu... more Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.
Biochemical and Biophysical Research Communications, Dec 15, 1988
We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors ... more We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors (IGFs) from BRL-3A cell conditioned medium. The fragment binds IGF-I and IGF-II with similar specificity to the 30 kDa binding protein, but with lower affinity. It corresponds to the carboxy terminus of the native binding protein (residues 148-270), and is thought to arise by proteolysis. We infer that this region of the native binding protein contains, at least in part, the IGF binding domain, o J.988 Academic P ..... Tnc.
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were as...
The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for pr... more The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% ...
Insulin-like growth factor II is a growth factor important in fetal development. Several cancer t... more Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observati...
Integrin receptors mediate the formation of adhesion complexes and play important roles in signal... more Integrin receptors mediate the formation of adhesion complexes and play important roles in signal transduction from the extracellular matrix. Integrin-based adhesion complexes (IAC) contain proteins that link integrins to the cytoskeleton and recruit signaling molecules, including vinculin, paxillin, focal adhesion kinase, talin and alpha-actinin. In this study, we describe a approximately 160 kDa protein that is markedly enriched at IAC induced by clustering integrins with fibronectin-coated beads. Protein sequence analysis reveals that this approximately 160 kDa protein is kinectin. Kinectin is an integral membrane protein found in endoplasmic reticulum, and it serves as a receptor for the motor protein kinesin. Fibronectin-induced IAC sequestered over half of the total cellular content of kinectin within 20 minutes. In addition, two other ER-resident proteins, RAP [low-density lipoprotein receptor-related protein (LRP) receptor-associated protein] and calreticulin, were found to ...
Current protocols in protein science / editorial board, John E. Coligan ... [et al.], 2011
Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are prese... more Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.
Proceedings of the National Academy of Sciences, 2006
RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of... more RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene-and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to
Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affe... more Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the a catalytic subunit of protein kinase CK2 (CK2a) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2a and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.
Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplast... more Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institutions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth muscle cell (SMC) proliferation, but do not effect endothelial cell (EC) proliferation. The purposes of this study were to (1) quantify the diffusive release of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) determine the effect of heparin and FGF-1 on SMC proliferation when the cells are immediately plated on the FG; and (3) by means of the diffusive release data, design a new in vitro model that may differentiate the effect of FG-incorporated FGF-1 and heparin, rather than the released, solubilized components of these two factors, on SMC and EC proliferation. 125I-FGF-1 or 3H-heparin release from FG into the overlying media was measured serially in a 96-hour period, either with or without cells. SMCs were immediately plated on FG containing various concentrations of FGF-1 and heparin. SMCs or ECs were plated on identical groups of FG containing FGF-1 and heparin 24 hours after the FG was made to exclude the effect on cell growth of the initial release of FGF-1 into the media. In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of the heparin in the FG was released into the overlying media, with minimal release occurring thereafter. The cell type or absence of cells did not affect release, but there was five times more FGF-1 and four times more heparin in the media at 72 hours for the immediate plating versus the delayed plating because of a diffusive release primarily in the first 24 hours. A heparin concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing immediate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, immediate plating induced greater proliferation than delayed plating; this was likely caused by the higher soluble FGF-1 concentration. Heparin doses as high as 500 U/mL had little effect on SMC proliferation. In contrast, ECs died with delayed plating on FG containing 500 U/mL heparin, and their growth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin. The differences in SMC proliferation when comparing immediate versus delayed plating are likely caused by diffusive release of heparin and FGF-1 into the media. Our ongoing work uses an optimized in vitro FG system that minimizes the effects of soluble factors. This is an important distinction, because the cytokines that are released in vivo will be removed by blood flow and, thus, may not exert an effect unless they are contained within the FG.
Background. The structure/function relationships of fibroblast growth factor 1 (FGF-1) are being ... more Background. The structure/function relationships of fibroblast growth factor 1 (FGF-1) are being investigated using site mutation, yielding novel structures with potential clinical applicability for modulating tissue responses to vascular interventions. We generated a mutant FGF-1 in which all three cysteines were converted to serines and then tested the relative mitogenic activities on endothelial cells (ECs) and smooth muscle cells (SMCs) and the molecular stability of the protein to thrombin-induced degradation.
The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angi... more The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-type HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity. Thus, the role of nuclear translocation in the mechanisms of action of HBGF-1 remains unclear.
The contribution of individual basic amino acids within three putative "consensus sequences" for ... more The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.
We have previously characterized the release of the signal peptide sequence-less fibroblast growt... more We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation.
In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (a... more In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (appMr) of 15,000 precursor form of human insulin-like growth factor-II (hIGF-II) was shown to be greater than that of completely processed hIGF-II for human fetal-derived fibroblasts, and both were more potent than rIGF-I. Since it is generally acknowledged that the stimulation of cell replication by the IGFs is mediated by IGF-I receptors, we undertook to determine whether differences between the receptors' affinity for the two Mr forms of hIGF-II and recombinant IGF-I (rIGF-I) or between its efficiency to couple specific growth factor occupancy to the activation of protein kinase could explain the greater replicating potential of appMr 15,000 hIGF-II. Equilibrium dissociation, i.e. Kd, and inhibition, i.e. Ki, constants were determined by measuring the ability of rIGF-I, hIGF-II, appMr 15,000 hIGF-II, insulin, and the antireceptor monoclonal antibody alpha IR-3 to compete with 125I-labeled rIGF-I and hIGF-II for binding to purified preparations of IGF-I receptors prepared from an enriched source of fetal membrane, i.e. human term placenta. The results of these experiments established that 1) hIGF-II and appMr 15,000 hIGF-II bind to the IGF-I receptor with the same affinity as rIGF-I, e.g. with Kd and Ki values between 0.03-0.07 nM; 2) the total binding capacity, i.e. Ro, for IGF-I binding was not statistically different from the Ro calculated for IGF-II binding; and 3) the statistical analysis of 12 data sets from the competitive binding experiments for goodness of fit indicated that a 1-site model for IGF-I and -II binding was a better fit of the data than a 2-site model. Measurements of the stimulation of IGF-I receptor autophosphorylation at low ligand concentrations established that appMr 15,000 hIGF-II and hIGF-II were more effective than rIGF-I in coupling receptor occupancy to the activation of its protein kinase. At saturating ligand concentrations, the 3 had similar potencies. The original preparation of appMr 15,000 hIGF-II contains a mixture of forms with acidic isoelectric points (pIs) and was more potent than Mr 7,500 IGF-II in stimulating receptor autophosphorylation. These results are consistent with the relative potencies of this preparation, hIGF-II, and rIGF-I in stimulating the replication of 12-week-old fetal dermal fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)
A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to hom... more A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.
Biochemical and Biophysical Research Communications, 1988
We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors ... more We have purified a 14 kDa fragment of the 30 kDa binding protein for insulin.like growth factors (IGFs) from BRL-3A cell conditioned medium. The fragment binds IGF-I and IGF-II with similar specificity to the 30 kDa binding protein, but with lower affinity. It corresponds to the carboxy terminus of the native binding protein (residues 148-270), and is thought to arise by proteolysis. We infer that this region of the native binding protein contains, at least in part, the IGF binding domain, o J.988 Academic P ..... Tnc.
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Papers by Brian Hampton