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A Yaron

Publications and source records attributed to A Yaron.

At least 19 recordsLinked to original sources
PubMed2001

Plexin-A3 mediates semaphorin signaling and regulates the development of hippocampal axonal projections.

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.

Animals↗
PubMed2001

Sorting of striatal and cortical interneurons regulated by semaphorin-neuropilin interactions.

Most striatal and cortical interneurons arise from the basal telencephalon, later segregating to their respective targets. Here, we show that migrating cortical interneurons avoid entering the striatum because of a chemorepulsive signal composed at least in part of semaphorin 3A and semaphorin 3F. Migrating interneurons expressing neuropilins, receptors for semaphorins, are directed to the cortex; those lacking them go to the striatum. Loss of neuropilin function increases the number of interneurons that migrate into the striatum. These observations reveal a mechanism by which neuropilins mediate sorting of distinct neuronal populations into different brain structures, and provide evidence that, in addition to guiding axons, these receptors also control neuronal migration in the central nervous system.

Animals↗
PubMed1998

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Amino Acid Sequence↗
PubMed1997

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Amino Acid Sequence↗
PubMed1995

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Adenosine Triphosphate↗
PubMed1995

Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells.

The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.

Antibodies↗
PubMed1995

In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Amino Acid Sequence↗
PubMed1994

Cholinotoxic effects on acetylcholinesterase gene expression are associated with brain-region specific alterations in G,C-rich transcripts.

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

Acetylcholinesterase↗
PubMed1994

Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV.

The addition of a soluble recombinant CD26 (sCD26) enhanced proliferation of peripheral blood lymphocytes induced by the recall antigen tetanus toxoid. sCD26 itself did not provide a mitogenic signal and did not augment the proliferative response of T cells to other mitogenic stimuli such as phytohemagglutinin and anti-CD3. Dipeptidyl peptidase IV-negative sCD26 did not have this enhancement effect, implying a requirement for enzyme activity. It was found that there exists a large variation in the levels of human plasma sCD26/dipeptidyl peptidase IV in vivo which may regulate T-cell activity. Peripheral blood lymphocytes from individuals whose plasma sCD26 was high and responded strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26. This suggests that plasma sCD26 had modulated the responsiveness of T cells of these individuals in vivo and that the endogenous plasma sCD26 regulates immune responses by allowing antigen-specific T cells to exert a maximal response to their specific antigen.

Adjuvants, Immunologic↗
PubMed1994

Focal scleral permeability enhanced by collagenase digestion. Experimental model of enzymatic sclerostomy.

PURPOSE: The purpose of this study is to demonstrate that restricted application of clostridial collagenase to the animal sclerolimbal area produces increased tissue permeability in vitro and results in a lasting significant decrease in the intraocular pressure (IOP) in vivo. METHODS: A special miniature device was developed to apply the carefully characterized enzyme to a small (1-mm diameter) and defined sclerolimbal area isolated from the surrounding tissue. The device was applied to the rabbit limbus for 4 hours and then removed. The enzyme was inactivated and washed out with an inhibitory solution. The IOP was measured by pneumotonometry, and the treated tissue was investigated by light and electron microscopy. Application and removal of the drug-delivery microapplicator was performed under topical anesthesia. RESULTS: Enzymatic degradation propagated without lateral spreading and reached at least two thirds of tissue width. Results of histologic examination of the treated area showed a thin tissue, characterized by degraded and disorganized collagen fibers, which were sharply separated from the intact fibers in the untreated area. This was sufficient to cause a tenfold increase in the basic transscleral flow in vitro, and a significant, relatively long-lasting (> 30 days) lowering of the mean IOP in 15 albino rabbits. In the first 10-day period after treatment, the IOP was reduced by 40%, and during the rest of the follow-up the IOP remained lowered by more than 20% (P > 0.0001) without any adjuvant treatment. CONCLUSION: Despite the small size of the collagenase-affected tissue, the evacuation rate of the aqueous humor was sufficient to cause a significant and relatively long-lasting lowering of IOP in rabbits. This mild treatment, performed under topical anesthesia, might present the basis for the development of an ambulatory treatment of human glaucoma.

Animals↗
PubMed1994

Site-directed mutagenesis of active site residues reveals plasticity of human butyrylcholinesterase in substrate and inhibitor interactions.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

Animals↗
PubMed1993

The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity.

T cells have been shown to express CD26, a known ectoenzyme with dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) activity in its extracellular domain. CD26 can also deliver a second costimulatory signal and contribute to T-cell activation. In an earlier study, we established CD26-transfected Jurkat T-cell lines and demonstrated that monoclonal antibody-mediated crosslinking of CD26 and CD3 induced interleukin 2 (IL-2) production. To determine the contribution of DPPIV enzymatic activity to the costimulatory activity of CD26, human CD26 cDNA was mutated so that active-site serine was replaced by alanine. The mutant CD26 antigen lacked DPPIV enzyme activity but still retained reactivity with three anti-CD26 monoclonal antibodies directed against distinct epitopes of CD26. After stimulation with a combination of anti-CD26 and anti-CD3 antibodies, wild-type CD26 (DPPIV+)-transfected Jurkat cells produced substantially more IL-2 than did mutant CD26 (DPPIV-) or CD26- control transfectants. Nevertheless, the mutant CD26-transfected cells still produced significantly more IL-2 than did CD26- control transfectants. These results suggest that DPPIV activity plays an important but not absolute role in the co-stimulatory activity of CD26 in this system. We also found that wild-type CD26 (DPPIV+) transfectants produced more IL-2 than mutant CD26 (DPPIV-)-transfected cells or CD26- control transfectants when triggered by stimuli not involving CD26, such as anti-CD3 and phorbol ester. These results suggest that the DPPIV activity of CD26 functions to augment the cellular responses of CD26-transfected Jurkat cells to external stimuli mediated by CD26 and/or the CD3/T-cell receptor complex, thus enhancing IL-2 production.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Regulation of insulin-like growth factor (IGF) binding protein-5 in the T47D human breast carcinoma cell line by IGF-I and retinoic acid.

The T47D human breast carcinoma cell line has been shown to synthesize insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) and IGF-I receptors, and to exhibit a mitogenic response to exogenous IGF-I. We have used T47D cells to investigate the regulation of IGFBPs by IGF-I and retinoic acid (RA), agents that affect cell proliferation and have been shown to regulate IGFBP levels in other cell types. Exposure of T47D cells to IGF-I resulted in the appearance of IGFBP-2, -4, and -5 in conditioned medium but had no effect on the levels of IGFBPs in Triton X-100-extracted cells. This effect was most pronounced for IGFBP-5 and was also elicited by an IGF-I analog that retains affinity for IGFBPs but not by insulin or IGF analogs that have decreased affinity for IGFBPs. Additionally, this effect was not associated with a change in IGFBP-5 messenger RNA (mRNA) levels; however, the appearance of IGFBP-5 in the conditioned medium was inhibited by an anti-IGF-I receptor antibody (alpha IR-3). RA decreased IGFBP-5 mRNA levels and cell-associated IGFBP-5 in both the presence and absence of IGF-I and inhibited the IGF-I-stimulated secretion of IGFBP-5 into T47D cell conditioned medium. These results suggest that IGF-I increases IGFBP-5 levels in the T47D cell line both through direct interaction with IGFBP-5 as well as through a receptor-mediated process that does not require direct interaction with IGFBPs. The latter results are consistent with an effect of IGF-I on a factor that may modulate an IGFBP protease activity. The inhibitory effect of RA, on the other hand, appears to be due primarily to regulation of IGFBP-5 mRNA levels. Thus, IGFBP-5 accumulation appears to be positively regulated by IGF-I, potentially at the level of susceptibility to proteolysis, and negatively regulated at the level of gene expression by RA.

Breast Neoplasms↗
PubMed1993

Proline-dependent structural and biological properties of peptides and proteins.

Proline residues confer unique structural constraints on peptide chains and markedly influence the susceptibility of proximal peptide bonds to protease activity. This review presents a critical analysis of peptidases involved in the cleavage of proline-containing peptide bonds, with particular attention to the role of proline peptidases in the regulation of the lifetime of biologically active peptides. Peptidases discussed include aminopeptidase P, prolidase, dipeptidyl peptidase IV, prolyl endopeptidase, and prolyl iminopeptidase. Attention is also given to HIV-1 protease, because this key enzyme processes an Xaa-Pro peptide bond. Analysis of the above enzymes reveals that they may function as key pacemakers in the control of the activity of many peptide hormones and that they are involved in a variety of immunological processes, including T-cell-mediated immune response. The novel occurrence of cis-trans isomerization about Xaa-Pro bonds and the biological function of peptidyl-prolyl cis-trans isomerases (immunophilins) are reviewed.

Amino Acid Sequence↗
PubMed1992

Aminopeptidase P from human leukocytes.

Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.

Amino Acid Sequence↗
PubMed1992

Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes.

The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.

Amino Acid Sequence↗
PubMed1992

Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P.

A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.

Aminopeptidases↗