N-terminal Tyrosine Residue Research Articles

Phospho-mimetic CD3ε variants prevent TCR and CAR signaling.

Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome. We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM. Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK. The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CARsignaling and highlights the key role of CD3ε signalosome to tune signal transduction.

Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

Tyrosine sulfation and O-glycosylation of chemoattractant receptor GPR15 differentially regulate interaction with GPR15L.

GPR15 is a G-protein-coupled receptor (GPCR) that directs lymphocyte homing to the colon and skin. Recent studies have identified a chemokine-like protein GPR15L (also known as C10orf99) as a functional ligand of GPR15. In this study, we examined the structural elements that regulate the GPR15-GPR15L interaction with primary focus on post-translational modifications (PTMs) of receptor N-terminus and on the C-terminus of the ligand. Our findings reveal that the GPR15 receptor is sulfated on the N-terminal tyrosine residue(s) and disruption of tyrosine sulfation inhibits binding of GPR15L. In contrast, the disruption of O-glycosylation on the N-terminal threonine or serine residues, or the removal of α2,3-linked sialic acids from O-glycans, enhances the GPR15L binding. Thus, GPR15 represents a unique chemoattractant receptor in which different N-terminal PTMs regulate its ligand binding in a contrasting manner. We further demonstrate that, unlike canonical chemokines, GPR15L activity critically requires its extreme C-terminal residue and that its hydrophobicity may be a key attribute that facilitates an optimal interaction with the receptor. Our results reveal novel insights into chemoattractant receptor-ligand interaction and provide a valid footing for potential intervention targeting the GPR15-GPR15L axis.

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in β-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.

Targeting G-quadruplex DNA with synthetic dendritic peptide: modulation of the proliferation of human cancer cells.

Telomerase, a reverse transcriptase enzyme, is found to over express in most cancer cells. It elongates the telomere region by repeated adding of TTAGGG in the 3′-end and leads to excess cell proliferation which causes cancer. G-quadruplex (G4) formation can inhibit such telomere lengthening. So, stabilization of G4 structure as well as inhibition of telomerase activity is very promising approach in targeted cancer therapy. Herein, the aptitude of a synthetic dendritic peptide, Cδ2–(YEE)–E (peptide 1), to target specifically the human telomeric G4 DNA, dAGGG(TTAGGG)3, has been evaluated. Both biochemical and biophysical techniques including gel mobility shift assay, isothermal titration calorimetry and fluorescence spectroscopy have been employed for the purpose. Circular dichroism study reveals that the targeting results an increase in thermal stability of G4 DNA. Interestingly, replacement of N-terminal tyrosine residue of peptide 1 by valine, Cδ2–(VEE)–E, (peptide 2) consequences in loss of its G4 DNA targeting ability, although both the peptides exhibit comparable affinity toward double-stranded DNA. Of note, peptide 1 causes cessation of growth of human cancer cells (HeLa and U2OS) and induces apoptosis in vitro. But it has no significant inhibitory effect on the growth of normal human embryonic kidney 293 cells. Mechanistically, Telomeric Repeat Amplification Protocol (TRAP) assay indicates that peptide 1 effectively inhibits the telomerase activity in human cell extracts. Overall, this study demonstrates the usefulness of a synthetic dendritic peptide as an inhibitor of tumor cell growth by inducing apoptosis upon targeting the telomeric G4 DNA.

Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3

Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.

Phosphoproteomic Analysis Reveals New SLP76 and PLCg1 Feedback Pathways That Regulate Lck in T Cells

Abstract T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. The exact mechanisms that regulate the spatial equilibrium of Lck between the TCR and downstream regulators are not well defined. While some of the important regulatory proteins involved in Lck feedback loops in T cells are also beginning to be defined, the pathways leading to the activation and recruitment of these inhibitory molecules are much more obscure. Modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways. Our recent data reveal a previously uncharacterized regulatory network where N-terminal tyrosine residues on SLP76 are involved in both positive and negative feedback loops, which regulate the phosphorylation of the activation loop on Lck. We also collected phosphoproteomic data that suggests that Lck targeting but not activity is regulated by PLCγ1. We examined the phosphoproteome of PLCγ1 deficient and reconstituted T cells through a timecourse of TCR stimulation. These data indicate that Lck activity was shifted from TCR to the SLP76 complex in the absence of PLCγ1. Altogether, wide-scale modern methods in quantitative mass spectrometry drive the molecular characterization of T cell signaling circuits and facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states.

A Gatekeeper Residue of ClpS1 from Arabidopsis thaliana Chloroplasts Determines its Affinity Towards Substrates of the Bacterial N-End Rule.

Proteins that are to be eliminated must be proficiently recognized by proteolytic systems so that inadvertent elimination of useful proteins is avoided. One mechanism to ensure proper recognition is the presence of N-terminal degradation signals (N-degrons) that are targeted by adaptor proteins (N-recognins). The members of the caseinolytic protease S (ClpS) family of N-recognins identify targets bearing an N-terminal phenylalanine, tyrosine, tryptophan or leucine residue, and then present them to a protease system. This process is known as the 'bacterial N-end rule'. The presence of a ClpS protein in Arabidopsis thaliana chloroplasts (AtClpS1) prompted the hypothesis that the bacterial N-end rule exists in this organelle. However, the specificity of AtClpS1 is unknown. Here we show that AtClpS1 has the ability to recognize bacterial N-degrons, albeit with low affinity. Recognition was assessed by the effect of purified AtClpS1 on the degradation of fluorescent variants bearing bacterial N-degrons. In many bacterial ClpS proteins, a methionine residue acts as a 'gatekeeper' residue, fine-tuning the specificity of the N-recognin. In plants, the amino acid at that position is an arginine. Replacement of this arginine for methionine in recombinant AtClpS1 allows for high-affinity binding to classical N-degrons of the bacterial N-end rule, suggesting that the arginine residue in the substrate-binding site may also act as a gatekeeper for plant substrates.

Oligomerization State of CXCL4 Chemokines Regulates G Protein-Coupled Receptor Activation.

CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first β-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.

Modeling of both shared and distinct interactions between MIF and its homologue D-DT with their common receptor CD74

D-dopachrome tautomerase (D-DT) shares amino acid sequence similarity, structural architecture and biological activity with the cytokine MIF. Recent studies show that the two protein homologs also bind to the same cell surface receptor, CD74, to activate the ERK1/2 pathway that ultimately leads to pro-inflammatory and pro-survival gene expression. We recently showed that RTL1000 and DRa1-MOG-35-55, two biological drugs with potent anti-inflammatory properties that treat experimental autoimmune encephalomyelitis (EAE) in mice, bind to the cell surface receptor CD74 with high affinity and compete with MIF for binding to the same regions of CD74. Computational modeling of MIF and RTL1000 binding interactions with CD74 predicted the presence of three CD74 binding regions for each MIF homotrimer. Through a similar approach we have now expanded our work to study the D-DT (MIF-2) interaction with CD74 that is mainly defined by three elements scattered throughout the disordered regions of the interacting molecules. The model predicted: (a) a hydrophobic cradle between CD74 and D-DT consisting of N-terminal tyrosine residues of three CD74 monomers arranged in a planar alignment interacts with aromatic amino acid residues located in the disordered D-DT C-terminus; (b) a triad consisting of the E103 residue on one D-DT monomer in close contact with R179 and S181 on one chain of the CD74 trimer forms an intermolecular salt bridge; and (c) amino acid residues on the C-terminus random coil of CD74 chain C form a long interacting area of ∼500Å2 with a disordered region of D-DT chain B. These three binding elements were also present in MIF/CD74 binding interactions, with involvement of identical or highly similar amino acid residues in each MIF homotrimer that partner with the exact same residues in CD74. Topologically, however, the location of the three CD74 binding regions of the D-DT homotrimer differs substantially from that of the three MIF binding regions. This key difference in orientation appears to derive from a sequence insertion in D-DT that topologically limits binding to only one CD74 molecule per D-DT homotrimer, in contrast to predicted binding of up to three CD74 molecules per MIF homotrimer. These results have implications for the manner in which D-DT and MIF compete with each other for binding to the CD74 receptor and for the relative potency of DRa1-MOG-35-55 and RTL1000 for competitive inhibition of D-DT and MIF binding and activation through CD74.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.

SLP76 N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal TCR signaling (TECH1P.848)

Abstract SLP76 is a cytosolic adaptor protein that plays an important role in TCR mediated T-cell signaling pathway. SLP76 consists of three functional domains: an amino-terminal acidic domain with three tyrosine phosphorylation sites, a central proline-rich domain, and a carboxy-terminal SH2 domain. The direct influence of each domain of SLP76 in T-cell signaling pathway is not well understood. In our work, phosphoproteomic methods in mass spectrometry were used to do a phosphoproteomic analysis of cells containing a mutant form of SLP76 where three tyrosine residues are replaced with phenylalanines. Thousands of unique phosphorylation sites upon a time course of T-cell receptor stimulation in these cells are identified. Mutation of the three N-terminal tyrosine residues of SLP76 phenocopied SLP76 deficient cells for the majority of tyrosine phosphorylation sites observed including feedback on proximal TCR signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when comparing Y3F mutant cells to SLP76 deficient cells. In addition, the N-terminal tyrosine sites of SLP76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397 and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine residues of SLP76 in T cell signaling and shed light on novel signaling networks that are uniquely regulated by SLP76 N terminal tyrosine residues.

Regulation of E3 ubiquitin ligase Cbl-b expression by protein tyrosine phosphatase SHP-1 (IRM10P.753)

Abstract Casitas B cell lymphoma protein-b (Cbl-b) is a RING finger family E3 ubiquitin ligase. It has been well documented that Cbl-b plays a crucial role in T cell activation and tolerance induction. Our recent study also indicates that Cbl-b regulates proallergic T cell development and allergic airway inflammation. Therefore, the regulation of Cbl-b expression in T cells is important for its biological functions. Our previous studies demonstrate that CD28 costimulation potentiates Cbl-b sel-ubiquitination, CTLA-4-B7 interaction induces Cbl-b expression. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b tyrosine phosphorylation in primary mouse T cells requires CD28 costimulation. The failure to detect Cbl-b tyrosine phosphorylation upon CD3 stimulation is not due to defective PTK activation, but rather interestingly SHP-1 is recruited to Cbl-b upon CD3 stimulation, while CD28 costimulation abrogates the interaction between Cbl-b and SHP-1. Furthermore, we identified Cbl-b N-terminal tyrosine residues within TKB domain and linker region (Y106, Y133, and Y363) are required for its E3 ubiquitin ligase activity. The aberrant Th2 phenotype observed in T cell-specific SHP-1-/- (tSHP-1-/-) mice is reminiscent of our recent observation that Th2 responses are heightened in Cbl-b-/- mice. In support of this notion, Cbl-b expression is down-regulated in naïve T cells of tSHP-1-/- mice.

Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-l-maurocalcine analog

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to 125I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10−7μl. 125I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of 125I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

The unsulfated extracellular N-terminus of vGPCR reduces the tumorigenicity of hGRO-α in nude mice

The Kaposi's Sarcoma-associated Herpesvirus (KSHV)-encoded G-protein coupled receptor (vGPCR) is an oncoprotein that is implicated in KSHV-associated malignancies. We previously revealed vGPCR incorporates sulfate groups within its extracellular N-terminal tyrosine residues (Y26 and Y28) and that this tyrosine sulfation is crucial for its tumorigenicity in nude mice. hGRO-α binds vGPCR in a sulfotyrosine-dependent manner and promotes its tumorigenicity through autocrine signaling. Interestingly, an unsulfated vGPCR mutant (yydd-vGPCR) attenuated the tumor growth triggered by hGRO-α. In this study, the extracellular N-terminus of vGPCR (wt-vGN) and an unsulfated vGPCR mutant (yydd-vGN) were individually secreted, expressed and purified. A radioactive labeling assay demonstrated that wt-vGN but not yydd-vGN incorporated [(35)S]-sulfate. In nude mice, NIH3T3 cells expressing yydd-vGN but not wt-vGN could significantly inhibit the tumor growth triggered by hGRO-α. All our data support the conclusion that the unsulfated extracellular N-terminus of vGPCR reduces the tumorigenicity of hGRO-α.

Factors that Restrict the Cell Permeation of Cyclic Prodrugs of an Opioid Peptide, Part 3: Synthesis of Analogs Designed to have Improved Stability to Oxidative Metabolism

Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.

Tyrosine Nitration within the Proline-Rich Region of Tau in Alzheimer's Disease

A substantial body of evidence suggests that nitrative injury contributes to neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative disorders. Previously, we showed in vitro that within the tau protein the N-terminal tyrosine residues (Y18 and Y29) are more susceptible to nitrative modifications than other tyrosine sites (Y197 and Y394). Using site-specific antibodies to nitrated tau at Y18 and Y29, we identified tau nitrated in both glial (Y18) and neuronal (Y29) tau pathologies. In this study, we report the characterization of two novel monoclonal antibodies, Tau-nY197 and Tau-nY394, recognizing tau nitrated at Y197 and Y394, respectively. By Western blot analysis, Tau-nY197 labeled soluble tau and insoluble paired helical filament proteins (PHF-tau) nitrated at Y197 from control and AD brain samples. Tau-nY394 failed to label soluble tau isolated from control or severe AD samples, but labeled insoluble PHF-tau to a limited extent. Immunohistochemical analysis using Tau-nY197 revealed the hallmark tau pathology associated with AD; Tau-nY394 did not detect any pathological lesions characteristic of the disorder. These data suggest that a subset of the hallmark pathological inclusions of AD contain tau nitrated at Y197. However, nitration at Y197 was also identified in soluble tau from all control samples, including those at Braak stage 0, suggesting that nitration at this site in the proline-rich region of tau may have normal biological functions in the human brain.

Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5′ LIC site of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino-oxy functionalized polyethylene glycol (PEG) ligand under aniline catalysis to provide a protein selectively modified at the N-terminus.

Sprouty Proteins Inhibit Receptor-mediated Activation of Phosphatidylinositol-specific Phospholipase C

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.

Balanced Vav2 GEF activity regulates neurite outgrowth and branching in vitro and in vivo

We have investigated the role of Vav2, a reported Rac1/Cdc42 GEF, on the development of Xenopus spinal neurons in vitro and in vivo. Both gain and loss of Vav2 function inhibited the rate neurite extension on laminin (LN), while only GFP-Vav2 over-expression enhanced process formation and branching. Vav2 over-expression protected neurons from RhoA-mediated growth cone collapse, similar to constitutively active Rac1, suggesting that Vav2 activates Rac1 in spinal neurons. Enhanced branching on LN required both Vav2 GEF activity and N-terminal tyrosine residues, but protection from RhoA-mediated collapse only required GEF activity. Interestingly, wild-type spinal neurons exhibited increased branching on the cell adhesion molecule L1, which required Vav2 GEF function, but not N-terminal tyrosine residues. Finally, we find that Vav2 differentially affects the Rohon–Beard peripheral and central process extension but promotes neurite branching of commissural interneurons near the ventral midline. Together, we suggest that balanced Vav2 activity is necessary for optimal neurite outgrowth and promotes branching by targeting GEF activity to branch points.