Functional definition of HLA-DR and -DQ epitopes specific for DR2-short,DwFJO haplotype

T-lymphocyte clones specific for the influenza A/Texas virus were obtained by limiting dilution of activated T cells from an HLA A2/3, B7/39, Cw -/-, DR2-short/2 short, DQw1/w1, DwFJO/FJO donor. Among the proliferating clones studied, and irrespective of their antigenic specificities, most of them were restricted by epitope(s) on HLA-DR molecules present only on DR2-short/DwFJO cells but not on DR2-negative or DR2-long positive (Dw2, Dw12, Dw-) cells. Two clones were restricted by epitopes borne by DQ products. Here again, these epitopes were present on DR2-short/DwFJO but not on DR2-long, DQw1 (Dw2, Dw12) cells, indicating that the DQwl molecules of DR2-long and DR2-short haplotypes are different. Taken together, these results indicate that the DR2-short, DwFJO haplotype is characterized by both HLA-DR- and DQ-specific molecules. Finally, one clone was restricted by an epitope shared by DR products from DR2 short/DwFJO, DRw11, and DRw13 haplotypes. This latter functional determinant has never been described until now.Abbreviations used in this paper APC antigen-presenting cells - HAU hemagglutinin units of influenza virus - HLA human leukocyte antigens - HTC homozygous typing cells - IL-2 interleukin 2 - mAb monoclonal antibody - MHC major histocompatibility complex - MLR mixed lymphocyte reactions - PBM peripheral blood mononuclear cells - %RR relative response percent     

A 320-kilobase artificial chromosome encoding the human HLA DR3-DQ2 MHC haplotype confers HLA restriction in transgenic mice

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact similar320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Abeta(null/null)) and expressing only murine CD4. A broad range of TCR Vbeta families was used in the peripheral T cell repertoire, which was also purged of Vbeta5-, Vbeta11-, and Vbeta12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Abeta(null/null) background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.     

Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

Redundancy in antigen-presenting function of the HLA-DR and -DQ molecules in the multiple sclerosis-associated HLA-DR2 haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.     

Increased risk of insulin-dependent diabetes for siblings of diabetic children with the DR 3 haplotype of the mother

Among 285 caucasoid families genotyped for HLA-A, B, C, DR including at least one insulin-dependent diabetic child, we have studied the effect of the DR3 and DR4 antigens inherited from the father or the mother (DR3p, DR3m, DR4p and DR4m, respectively) on the recurrence of the disease among siblings; families with affected parents being excluded, a total of 37 affected and 200 non affected siblings have been taken into consideration. Among the DR3, DR4 positive siblings, the DR4p/DR3m genotype was observed at a greater frequency than the DR3p/DR4m genotype among affected, but not among unaffected siblings. Comparing the respective frequencies between affected and unaffected siblings, the relative risk was 8.1 (p less than 10(-6) among DR4p/DR3m positive siblings, but is was not significantly increased among DR3p/DR4m positive siblings. The excess of maternal DR3 among affected siblings of diabetic children could be due to a gestational event associated with HLA-DR3, e.g. education of the fetal immune repertoire or the transmission of a viral infection by the mother to the fetus during pregnancy, after reactivation of the latent viral disease.     

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.     

CCL2 and CCL3 are essential mediators of pelvic pain in experimental autoimmune prostatitis

Experimental autoimmune prostatitis (EAP) is a murine model of chronic prostatitis/chronic pelvic pain syndrome (CPPS) in men, a syndrome characterized by chronic pelvic pain. We have demonstrated that chemokine ligands CCL2 and CCL3 are biomarkers that correlate with pelvic pain symptoms. We postulated that CCL2 and CCL3 play a functional role in CPPS and therefore examined their expression in EAP. Upon examination of the prostate 5 days after induction of EAP, CCL2 mRNA was elevated 2- to 3-fold, CCL8 by 15-fold, CCL12 by 12- to 13-fold, and CXCL9 by 2- to 4-fold compared with control mice. At 10 days the major chemokines were CXCL13 and CXCL2; at 20 days CCL2 (1- to 2-fold), CCL3 (2- to 3-fold) and CCL11 (2- to 3-fold); and at 30 days, CCL12 (20- to 35-fold) and smaller increases in CCL2, CCL3, and XCL1. Chemokine elevations were accompanied by increases in mast cells and B cells at 5 days, monocytes and neutrophils at day 10, CD4+ T cells at day 20, and CD4+ and CD8+ T cells at day 30. Anti-CCL2 and anti-CCL3 neutralizing antibodies administered at EAP onset attenuated pelvic pain development, but only anti-CCL2 antibodies were effective therapeutically. CCL2- and its cognate receptor CCR2-deficient mice were completely protected from development of pain symptoms but assumed susceptibility after reconstitution with wild-type bone marrow. CCL3-deficient mice showed resistance to the maintenance of pelvic pain while CCR5-deficient mice did not show any lessening of pelvic pain severity. These results suggest that the CCL2-CCR2 axis and CCL3 are important mediators of chronic pelvic pain in EAP.     

Synthesis and immunological characterization of two peptides which are models for two of the four major antigenic sites of a scorpion toxin

We have synthesized, by the solid phase procedure, then purified and chemically characterized two peptides. They mimic two regions of toxin II of the scorpion Androctonus australis Hector, one around disulfide bridge 12-63 and another at sequence 50-59. Each of these two regions was supposed to include an antigenic site. We have shown that the synthetic replicas of these regions are individually recognized by a part of IgGs raised against native toxin II. This is a strong argument for the involvement of these areas in the antigenicity of the toxin. Furthermore, region-specific IgGs purified by affinity chromatography on the two Sepharose linked peptides were able to bind 125I-labelled toxin II.     

Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.     

Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence psiKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.     

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.     

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.     

Synthesis and characterization of cyclic peptides that are β‐helical in trifluoroethanol

We show that three designed cyclic d ,l ‐peptides are β‐helical in TFE—a solvent in which the archetypal β‐helical peptide, gA, is unstructured. This result represents an advance in the field of β‐helical peptide foldamers and a step toward achieving β‐helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG–PS composite resin prefunctionalized with the alkanesulfonamide ‘SCL’ linker and preloaded with glycine. Our new conditions avoided C‐terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic β‐helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Transport characteristics of peptides and peptidomimetics: I. N-methylated peptides as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa.

Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N-Methylation has been used successfully in peptide-based drug design in an attempt to improve the metabolic stability of a peptide-based lead compound. However, the effect of N-methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P-glycoprotein (P-gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N-acetylated and C-amidated) analogs of H-Ala-Phe-Ala-OH with N-methyl groups on either the Ala-Phe or Phe-Ala peptide bond were synthesized. Transport studies using Caco-2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N-methylation of the Ala-Phe peptide bond of H-Ala-Phe-Ala-OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N-methylation of the Phe-Ala peptide bond of H-Ala-Phe-Ala-OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N-methylation of the Phe-Ala peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 decreased the substrate activity of the molecule for the efflux transporter P-gp. In contrast, N-methylation of the Ala-Phe peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 increased the substrate activity of the molecule for P-gp.     

Endogenous oocyte antigens are required for rapid induction and progression of autoimmune ovarian disease following day-3 thymectomy

Female (C57BL/6xA/J)F(1) mice undergoing thymectomy on day 3 after birth (d3tx) developed autoimmune ovarian disease (AOD) and autoimmune disease of the lacrimal gland. As both were prevented by normal adult CD25(+) T cells, regulatory T cell depletion is responsible for d3tx diseases. AOD began as oophoritis at 3 wk. By 4 wk, AOD progressed to ovarian atrophy with autoantibody response against multiple oocyte Ag of early ontogeny. The requirement for immunogenic endogenous ovarian Ag was investigated in d3tx female mice, d3tx male mice, and d3tx neonatally ovariectomized (OX) females. At 8 wk, all mice had comparable lacrimalitis but only those with endogenous ovaries developed AOD in ovarian grafts. The duration of Ag exposure required to initiate AOD was evaluated in d3tx mice OX at 2, 3, or 4 wk and engrafted with an ovary at 4, 5, or 6 wk, respectively. The mice OX at 2 wk did not have oophoritis whereas approximately 80% of mice OX at 3 or 4 wk had maximal AOD, thus Ag stimulus for 2.5 wk following d3tx is sufficient. AOD progression requires additional endogenous Ag stimulation from the ovarian graft. In mice OX at 3 wk, ovaries engrafted at 5 wk had more severe oophoritis than ovaries engrafted at 6 or 12 wk; moreover, only mice engrafted at 5 wk developed ovarian atrophy and oocyte autoantibodies. Similar results were obtained in mice OX at 4 wk. Thus endogenous tissue Ag are critical in autoimmune disease induction and progression that occur spontaneously upon regulatory T cell depletion.     

eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase

Class-I phosphoinositide 3-kinases (PI 3-kinases) are dual specificity enzymes that possess both lipid and protein kinase activity. While the best characterized property of this protein kinase is as an autokinase activity, there have also been reports it can phosphorylate exogenous substrates including peptides, IRS-1 and PDE-3B. The identification of two novel potential protein substrates of PI 3-kinase is described here. By employing in vitro kinase assays using recombinant proteins as the substrates, it is shown that the translational regulator 4EBP1 becomes phosphorylated by the p110alpha and p110gamma isoforms of class-I PI 3-kinases. The lipid kinase activity of both these isoforms is increased by allosteric binding of H-Ras or betagamma subunits of heterotrimeric G proteins, but we find this is not the case for the protein kinase activity. Surprisingly though, a site on H-Ras is phosphorylated by p110alpha and p110gamma. This raises the possibility that these proteins could serve as physiological substrates for the protein kinase activity of PI 3-kinase and suggests this activity operates in a physiological context by phosphorylating substrates other than the PI 3-kinase itself. This may be particularly important in regulating the interaction of Ras with PI 3-kinase.