Publications by authors named "Irwan T Makagiansar"

11 Publications

  • Page 1 of 1

Suppression of type 1 diabetes in NOD mice by bifunctional peptide inhibitor: modulation of the immunological synapse formation.

Chem Biol Drug Des 2007 Sep;70(3):227-36

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA.

The aim of this work was to design and utilize a bifunctional peptide inhibitor called glutamic acid decarboxylase-bifunctional peptide inhibitor to suppress the progression of type 1 diabetes in non-obese diabetic mice. The hypothesis is that glutamic acid decarboxylase-bifunctional peptide inhibitor binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule type 1 on antigen-presenting cell and inhibits the immunological synapse formation during T-cell-antigen-presenting cell interactions. Glutamic acid decarboxylase-bifunctional peptide inhibitor was composed of a major epitope of the type 1 diabetes-associated antigen, glutamic acid decarboxylase 65 kDa, covalently linked to a peptide derived from CD11a of lymphocyte function-associated antigen-1. The suppression of insulitis and type 1 diabetes was evaluated using non-obese diabetic and non-obese diabetic severe combined immunodeficiency mice. Glutamic acid decarboxylase-bifunctional peptide inhibitor had the capacity to suppress invasive insulitis in non-obese diabetic mice. CD4+ T-cells isolated from glutamic acid decarboxylase-bifunctional peptide inhibitor treated mice also suppressed insulitis and hyperglycemia when transferred with diabetogenic non-obese diabetic spleen cells into non-obese diabetic severe combined immunodeficiency recipients. As predicted, the glutamic acid decarboxylase-bifunctional peptide inhibitor cross-linked a significant fraction of major histocompatibility complex class-II molecules to intercellular adhesion molecule type 1 molecules on the surface of live antigen-presenting cell. Intravenous injection of the glutamic acid decarboxylase-bifunctional peptide inhibitor elicited interleukin-4-producing T-cells in non-obese diabetic mice primed against the glutamic acid decarboxylase-epitope peptide. Together, the results indicate that glutamic acid decarboxylase-bifunctional peptide inhibitor induces interleukin-4-producing regulatory cells but does not expand the glutamic acid decarboxylase-specific Th2 population. Given that Th2 effector cells can cause pathology, the glutamic acid decarboxylase-bifunctional peptide inhibitor may represent a novel mechanism to induce interleukin-4 without Th2-associated pathology.
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http://dx.doi.org/10.1111/j.1747-0285.2007.00552.xDOI Listing
September 2007

Differential phosphorylation of NG2 proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration.

J Cell Biol 2007 Jul 25;178(1):155-65. Epub 2007 Jun 25.

Cancer Center, The Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

Two distinct Thr phosphorylation events within the cytoplasmic domain of the NG2 proteoglycan help regulate the cellular balance between proliferation and motility. Protein kinase Calpha mediates the phosphorylation of NG2 at Thr2256, resulting in enhanced cell motility. Extracellular signal-regulated kinase phosphorylates NG2 at Thr2314, stimulating cell proliferation. The effects of NG2 phosphorylation on proliferation and motility are dependent on beta1-integrin activation. Differential cell surface localization of the two distinctly phosphorylated forms of NG2 may be the mechanism by which the NG2-beta1-integrin interaction promotes proliferation in one case and motility in the other. NG2 phosphorylated at Thr2314 colocalizes with beta1-integrin on microprotrusions from the apical cell surface. In contrast, NG2 phosphorylated at Thr2256 colocalizes with beta1-integrin on lamellipodia at the leading edges of cells. Thus, phosphorylation and the resulting site of NG2-integrin localization may determine the specific downstream effects of integrin signaling.
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http://dx.doi.org/10.1083/jcb.200612084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064431PMC
July 2007

Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells.

J Cell Sci 2006 Jun 23;119(Pt 12):2457-67. Epub 2006 May 23.

Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA.

Human cytomegalovirus (HCMV) infection results in endothelial dysfunction, typically known as dysregulated apoptosis, and aberrant expression and sub-cellular localization of p53, a tumor suppressor that accumulates at the late stage of infection. In this study, we examined three hypotheses that could be responsible for HCMV-induced cytoplasmic p53 accumulation at the later stage of infection: hyperactive nuclear export, cytoplasmic p53 tethering and delayed p53 degradation. Leptomycin B treatment, a nuclear export inhibitor, was unable to reduce cytoplasmic p53, thereby eliminating the hyperactive nuclear export mechanism. The findings that nascent p53 still entered nuclei after the nuclear export inhibition indicated that cytoplasmic tethering may play a minor role. Cytoplasmic p53 was still observed after the translation activities were blocked by cycloheximide. There was more than an eight-fold increase in the cytoplasmic p53 half-life with abnormal p53 ubiquitination. Taken together, these results suggest that delayed degradation could be responsible for the cytoplasmic p53 accumulation. The general slow-down of the proteasomal activity and the dysregulated p53 ubiquitination process at the later stage of infection could contribute to the reduced cytoplasmic p53 degradation and might be relevant to dysregulated endothelial apoptosis. The HCMV-induced changes in p53 dynamics could contribute to endothelial dysfunction.
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http://dx.doi.org/10.1242/jcs.02974DOI Listing
June 2006

Molecular basis of interaction between NG2 proteoglycan and galectin-3.

J Cell Biochem 2006 May;98(1):115-27

Burnham Institute, Developmental Neurobiology Program, La Jolla, California 92037, USA.

Previous work has demonstrated the ability of the NG2 proteoglycan, a component of microvascular pericytes, to stimulate endothelial cell motility and morphogenesis. This function of NG2 depends on formation of a complex with galectin-3 and alpha3beta1 integrin to stimulate integrin-mediated transmembrane signaling. In addition, the co-expression of galectin-3 and NG2 in A375 melanoma cells suggests that the malignant properties of these cells may be affected by interaction between the two molecules. Here, we extend the theme of co-expression and interaction of NG2 and galectin-3 to human glioma cells. We also establish a molecular basis for the NG2/galectin-3 interaction. The C-terminal carbohydrate recognition domain of galectin-3 is responsible for binding to the NG2 core protein. Within the NG2 extracellular domain, the membrane-proximal D3 segment of the proteoglycan contains the primary binding site for interaction with galectin-3. The interaction between galectin-3 and NG2 is a carbohydrate-dependent one mediated by N-linked rather than O-linked oligosaccharides within the D3 domain of the NG2 core protein. These studies establish a foundation for attempts to reduce the aggressive properties of tumor cells by disrupting the NG2/galectin-3 interaction.
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http://dx.doi.org/10.1002/jcb.20768DOI Listing
May 2006

Phosphorylation of NG2 proteoglycan by protein kinase C-alpha regulates polarized membrane distribution and cell motility.

J Biol Chem 2004 Dec 25;279(53):55262-70. Epub 2004 Oct 25.

Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

Protein kinase C (PKC)-alpha phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr(2256), identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and alpha(3)beta(1) integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-alpha-mediated NG2 phosphorylation at Thr(2256) is therefore a key step for initiating cell polarization and motility.
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http://dx.doi.org/10.1074/jbc.M411045200DOI Listing
December 2004

NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin.

Mol Biol Cell 2004 Aug 4;15(8):3580-90. Epub 2004 Jun 4.

The Burnham Institute, La Jolla, California 92037, USA.

The NG2 proteoglycan is expressed by microvascular pericytes in newly formed blood vessels. We have used in vitro and in vivo models to investigate the role of NG2 in cross-talk between pericytes and endothelial cells (EC). Binding of soluble NG2 to the EC surface induces cell motility and multicellular network formation in vitro and stimulates corneal angiogenesis in vivo. Biochemical data demonstrate the involvement of both galectin-3 and alpha3beta1 integrin in the EC response to NG2 and show that NG2, galectin-3, and alpha3beta1 form a complex on the cell surface. Transmembrane signaling via alpha3beta1 is responsible for EC motility and morphogenesis in this system. Galectin-3-dependent oligomerization may potentiate NG2-mediated activation of alpha3beta1. In conjunction with recent studies demonstrating the early involvement of pericytes in angiogenesis, these data suggest that pericyte-derived NG2 is an important factor in promoting EC migration and morphogenesis during the early stages of neovascularization.
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http://dx.doi.org/10.1091/mbc.e04-03-0236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC491820PMC
August 2004

Expression, purification, and structural study of the EC4 domain of E-cadherin.

Protein Expr Purif 2004 Jan;33(1):72-9

Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA.

The objective of this work was to produce unlabeled and 15N-labeled EC4 domain protein from E-cadherin for studying its structure and binding properties to other EC domains as well as to E-cadherin peptides. The EC4 domain of E-cadherin was expressed in Escherichia coli from the vector pASK-IBA6 and localized in the periplasmic space of E. coli. This protein contains a Streptag sequence at the N-terminus, and thus was purified using a Strep-Tactin affinity column. However, at high concentrations the 15N-labeled EC4 protein showed an unstable conformation. Conditions for stabilizing the conformation of this protein were evaluated using CD spectroscopy. The CD results showed that this protein has high conformational stability in Tris buffer at pH 6.0 in the presence of 10 mM calcium chloride.
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http://dx.doi.org/10.1016/j.pep.2003.08.021DOI Listing
January 2004

Localized production of human E-cadherin-derived first repeat in Escherichia coli.

Protein Expr Purif 2002 Dec;26(3):449-54

Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047-2504, USA.

E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.
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http://dx.doi.org/10.1016/s1046-5928(02)00553-3DOI Listing
December 2002

Increasing paracellular porosity by E-cadherin peptides: discovery of bulge and groove regions in the EC1-domain of E-cadherin.

Pharm Res 2002 Aug;19(8):1170-9

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.

Purpose: The objective of this work is to evaluate the ability of peptides derived from the bulge (HAV-peptides) and groove (ADT-peptides) regions of E-cadherin EC1-domain to increase the paracellular porosity of the intercellular junctions of Madin-Darby canine kidney (MDCK) cell monolayers.

Methods: Peptides were synthesized using a solid-phase method and were purified using semi-preparative HPLC. MDCK monolayers were used to evaluate the ability of cadherin peptides to modulate cadherin-cadherin interactions in the intercellular junctions. The increase in intercellular junction porosity was determined by the change in transepithelial electrical resistance (TEER) values and the paracellular transport of 14C-mannitol.

Results: HAV- and ADT-peptides can lower the TEER value of MDCK cell monolayers and enhance the paracellular permeation of 14C-mannitol. HAV- and ADT-decapeptides can modulate the intercellular junctions when they are added from the basolateral side but not from the apical side; on the other hand. HAV- and ADT-hexapeptides increase the paracellular porosity of the monolayers when added from either side. Conjugation of HAV- and ADT-peptides using omega-aminocaproic acid can only work to modulate the paracellular porosity when ADT-peptide is at the N-terminus and HAV-peptide is at the C-terminus; because of its size, the conjugate can only modulate the intercellular junction when added from the basolateral side.

Conclusions: Peptides from the bulge and groove regions of the EC1 domain of E-cadherin can inhibit cadherin-cadherin interactions, resulting in the opening of the paracellular junctions. These peptides may be used to improve paracellular permeation of peptides and proteins. Furthermore, this work suggests that both groove and bulge regions of EC-domain are important for cadherin-cadherin interactions.
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http://dx.doi.org/10.1023/a:1019850226631DOI Listing
August 2002

N-cadherin involvement in the heterotypic adherence of malignant T-cells to epithelia.

Mol Cell Biochem 2002 Apr;233(1-2):1-8

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.

N-cadherin, a cell adhesion molecule normally found in neural cell tissue, has been found recently to be expressed on the surface of malignant T-cells. The function of N-cadherin on these cells remains unclear. Heterotypic assays between Molt-3 T lymphoblastic leukemia cells and Caco-2 epithelial monolayers were examined under different conditions to assess the functional role of N-cadherin. The results indicate that adherence of Molt-3 cells to Caco-2 monolayers was reduced significantly following pretreatment of Molt-3 cells with 100 microM of an N-cadherin-derived antagonist decapeptide. In contrast, pretreatment of Molt-3 cells with an anti-N-cadherin antibody raised against the first 20 amino acids of N-cadherin sequence led to a surprisingly marked enhancement of Molt-3 cell adherence to Caco-2 monolayers. In addition, the presence of anti-N-cadherin antibody neutralized the inhibitory effect of anti-ICAM-1 on Molt-3 adhesion to Caco-2 monolayers. This novel finding demonstrates that external stimulus through the N-cadherin amino terminus can modulate adhesion of malignant T-cells to epithelia and may promote their ability to invade or metastasize to inflammatory sites.
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http://dx.doi.org/10.1023/a:1015556625038DOI Listing
April 2002

Disulfide bond formation promotes the cis- and trans-dimerization of the E-cadherin-derived first repeat.

J Biol Chem 2002 May 20;277(18):16002-10. Epub 2002 Feb 20.

Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.

Cadherin is a cell adhesion molecule crucial for epithelial and endothelial cell monolayer integrity. The previously solved x-ray crystallographic structure of the E-CAD12 cis-dimer displayed an unpaired Cys(9), which protruded away from the Cys(9) on the other protomer. To investigate the possible biological function of Cys(9) within the first repeat (the E-cadherin-derived N-terminal repeat), E-CAD1 was overexpressed and secreted into the periplasmic space of Escherichia coli cells. Recombinant E-CAD1 produced a mixed monomer and dimer in an equilibrium fashion. The dimer was linked by a disulfide through Cys(9) pairing. Analysis by high pressure liquid chromatography and electron microscopy suggested the existence of oligomeric complexes. Mutation at Trp(2) appears to indicate that these oligomeric complexes trans-dimerize. Interestingly, mutation of Cys(9) affected not only the cis-dimerization, but also the trans-oligomerization of E-CAD1. Accordingly, it is plausible that, under oxidative stress, the homophilic interactions of E-cadherin through E-CAD1 may be promoted and stabilized by this disulfide bond.
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http://dx.doi.org/10.1074/jbc.M200916200DOI Listing
May 2002