Peptide synthesis using unprotected peptides through orthogonal coupling methods.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.

A potent inhibitor of endothelial cell proliferation is generated by proteolytic cleavage of the chemokine platelet factor 4.

Platelet factor 4 (PF-4) is an archetype of the "chemokine" family of low molecular weight proteins that play an important role in injury responses and inflammation. From activated human leukocyte culture supernatants, we have isolated a form of PF-4 that acts as a potent inhibitor of endothelial cell proliferation. The PF-4 derivative is generated by peptide bond cleavage between Thr-16 and Ser-17, a site located downstream from the highly conserved and structurally important CXC motif. The unique cleavage leads to a loss of one of the structurally important large loops in the PF-4 molecule and generation of an N terminus with basic residues that have the potential to interact with the acidic extracellular domain of the G-protein-coupled chemokine receptor. The N-terminal processed PF-4 exhibited a 30- to 50-fold greater growth inhibitory activity on endothelial cells than PF-4. Since endothelial cell growth inhibition is the only known cellular activity of the cleaved PF-4, we have designated this chemokine endothelial cell growth inhibitor. The N-terminal processing of PF-4 may represent an important mechanism for modulating PF-4 activity on endothelial cells during tissue injury, inflammation, and neoplasia.

Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat.

Our previous studies have shown that stimulation of the anterior ventral third ventricular region increases atrial natriuretic peptide (ANP) release, whereas lesions of this structure, the median eminence, or removal of the neural lobe of the pituitary block ANP release induced by blood volume expansion (BVE). These results indicate that participation of the central nervous system is crucial in these responses, possibly through mediation by neurohypophysial hormones. In the present research we investigated the possible role of oxytocin, one of the two principal neurohypophysial hormones, in the mediation of ANP release. Oxytocin (1-10 nmol) injected i.p. caused significant, dose-dependent increases in urinary osmolality, natriuresis, and kaliuresis. A delayed antidiuretic effect was also observed. Plasma ANP concentrations increased nearly 4-fold (P < 0.01) 20 min after i.p. oxytocin (10 nmol), but there was no change in plasma ANP values in control rats. When oxytocin (1 or 10 nmol) was injected i.v., it also induced a dose-related increase in plasma ANP at 5 min (P < 0.001). BVE by intra-atrial injection of isotonic saline induced a rapid (5 min postinjection) increase in plasma oxytocin and ANP concentrations and a concomitant decrease in plasma arginine vasopressin concentration. Results were similar with hypertonic volume expansion, except that this induced a transient (5 min) increase in plasma arginine vasopressin. The findings are consistent with the hypothesis that baroreceptor activation of the central nervous system by BVE stimulates the release of oxytocin from the neurohypophysis. This oxytocin then circulates to the right atrium to induce release of ANP, which circulates to the kidney and induces natriuresis and diuresis, which restore body fluid volume to normal levels.

Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39.

PR-39 is a porcine 39-aa peptide antibiotic composed of 49% proline and 24% arginine, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis. PR-39 was originally isolated from pig small intestine, but subsequent cDNA cloning showed that the gene is expressed in the bone marrow. The open reading frame of the clone showed that PR-39 is made as 173-aa precursor whose proregion belongs to the cathelin family. The PR39 gene, which is rather compact and spans only 1784 bp has now been sequenced. The coding information is split into four exons. The first exon contains the signal sequence of 29 residues and the first 37 residues of the cathelin propart. Exons 2 and 3 contain only cathelin information, while exon 4 codes for the four C-terminal cathelin residues and the mature PR-39 peptide extended by three residues. The sequenced upstream region (1183 bp) contains four potential recognition sites for NF-IL6 and three for APRF, transcription factors known to regulate genes for both cytokines and acute phase response factors. Genomic hybridizations revealed a fairly high level of restriction fragment length polymorphism and indicated that there are at least two copies of the PR39 gene in the pig genome. PR39 was mapped to pig chromosome 13 by linkage and in situ hybridization mapping. The gene for the human peptide antibiotic FALL-39 (also a member of the cathelin family) was mapped to human chromosome 3, which is homologous to pig chromosome 13.

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

Valutazione della risposta immunitaria cellulare e fenotipizzazione linfocitaria nel suino in corso di infezione naturale da virus della PRRS (“Porcine Reproductive and Respiratory Syndrome”) e dopo stimolazione di PBMC (“Peripheral Blood Mononuclear Cells”) in vitro con Peptide Killer (KP)

SUMMARY The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is one of the most spread pathogens in swine herds all over the world and responsible for a reproductive and respiratory syndrome that causes severe heath and economical problems. This virus emerged in late 1980’s but although about 30 years have passed by, the knowledge about some essential facets related to the features of the virus (pathogenesis, immune response, and epidemiology) seems to be still incomplete. Taking into account that the development of modern vaccines is based on how innate and acquire immunity react, a more and more thorough knowledge on the immune system is needed, in terms of molecular modulation/regulation of the inflammatory and immune response upon PRRSV infection. The present doctoral thesis, which is divided into 3 different studies, is aimed to increase the knowledge about the interaction between the immune system and the PRRS virus upon natural infection. The objective of the first study entitled “Coordinated immune response of memory and cytotoxic T cells together with IFN-γ secreting cells after porcine reproductive and respiratory syndrome virus (PRRSV) natural infection in conventional pigs” was to evaluate the activation and modulation of the immune response in pigs naturally infected by PRRSV compared to an uninfected control group. The course of viremia was evaluated by PCR, the antibody titres by ELISA, the number of IFN-γ secreting cells (IFN- SC) by an ELISPOT assay and the immunophenotyping of some lymphocyte subsets (cytotoxic cells, memory T lymphocytes and cytotoxic T lymphocytes) by flow cytometry. The results showed that the activation of the cell-mediated immune response against PRRSV is delayed upon infection and that however the levels of IFN-γ SC and lymphocyte subsets subsequently increase over time. Furthermore, it was observed that the course of the different immune cell subsets is time-associated with the levels of PRRSV-specific IFN-γ SC and this can be interpreted based on the functional role that such lymphocyte subsets could have in the specific production/secretion of the immunostimulatory cytokine IFN-γ. In addition, these data support the hypothesis that the age of the animals upon the onset of infection or the diverse immunobiological features of the field isolate, as typically hypothesized during PRRSV infection, are critical conditions able to influence the qualitative and quantitative course of the cell-mediated immune response during PRRSV natural infection. The second study entitled “Immune response to PCV2 vaccination in PRRSV viremic piglets” was aimed to evaluate whether PRRSV could interfere with the activation of the immune response to PCV2 vaccination in pigs. In this trial, 200 pigs were divided into 2 groups: PCV2-vaccinated (at 4 weeks of age) and PCV2-unvaccinated (control group). Some piglets of both groups got infected by PRRSV, as determined by PRRSV viremia detection, so that 4 groups were defined as follows: PCV2 vaccinated - PRRSV viremic PCV2 vaccinated - PRRSV non viremic PCV2 unvaccinated - PRRSV viremic PCV2 unvaccinated - PRRSV non viremic The following parameters were evaluated in the 4 groups: number of PCV2-specific IFN-γ secreting cells, antibody titres by ELISA and IPMA. Based on the immunological data analysis, it can be deduced that: 1) The low levels of antibodies against PCV2 in the PCV2-vaccinated – PRRSV-viremic group at vaccination (4 weeks of age) could be related to a reduced colostrum intake influenced by PRRSV viremia. 2) Independently of the viremia status, serological data of the PCV2-vaccinated group by ELISA and IPMA does not show statistically different differences. Consequently, it can be be stated that, under the conditions of the study, PRRSV does not interfere with the antibody response induced by the PCV2 vaccine. 3) The cell-mediated immune response in terms of number of PCV2-specific IFN-γ secreting cells in the PCV2-vaccinated – PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN-γ secreting cells after PCV2 vaccination, compared to the PCV2-vaccinated – PRRSV-non-viremic group. The data highlight and further support the inhibitory role of PRRSV on the development and activation of the immune response and highlight how a natural infection at early age can negatively influence the immune response to other pathogens/antigens. The third study entitled “Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8αβ+ T cell subsets by an antibody-derived killer peptide (KP)” was aimed to determine whether and how the killer peptide (KP) could modulate the immune response in terms of activation of specific lymphocyte subsets. This is a preliminary approach also aimed to subsequently evaluate such KP with a potential antivural role or as adjuvant. In this work, pig peripheral blood mononuclear cells (PBMC) were stimulated with three KP concentrations (10, 20 and 40 g/ml) for three time points (24, 48 and 72 hours). TIME POINTS (hours) KP CONCENTRATIONS (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 By using flow cytometry, the qualitative and quantitative modulation of the following immune subsets was evaluated upon KP stimulation: monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and CD4+ and CD8α/β+ T lymphocyte subsets. Based on the data, it can be deduced that: 1) KP promotes a dose-dependent activation of monocytes, particularly after 24 hours of stimulation, by inducing a monocyte phenotypic and maturation shift mainly involved in sustaining the innate/inflammatory response. 2) KP induces a strong dose-dependent modulation of NK and NKT cells, characterized by an intense increase of the NKT cell fraction compared to NK cells, both subsets involved in the antibody-dependent cell cytotoxicity (ADCC). The increase is observed especially after 24 hours of stimulation. 3) KP promotes a significant activation of the cytotoxic T lymphocyte subset (CTL). 4) KP can modulate both the T helper and T cytotoxic phenotype, by inducing T helper cells to acquire the CD8α thus becoming doube positive cells (CD4+CD8+) and by inducing CTL (CD4-CD8+high) to acquire the double positive phenotype (CD4+CD8α+high). Therefore, KP may induce several effects on different immune cell subsets. For this reason, further research is needed aimed at characterizing each “effect” of KP and thus identifying the best use of the decapeptide for vaccination practice, therapeutic purposes or as vaccine adjuvant. RIASSUNTO Il virus della PRRS (Porcine Reproductive Respiratory Syndrome) è uno dei più diffusi agenti patogeni negli allevamenti suini di tutto il mondo, responsabile di una sindrome riproduttiva e respiratoria causa di gravi danni ad impatto sanitario ed economico. Questo virus è emerso attorno alla fine degli anni ’80 ma nonostante siano passati circa una trentina di anni, le conoscenze su alcuni punti essenziali che riguardano le caratteristiche del virus (patogenesi, risposta immunitaria, epidemiologia) appaiono ancora spesso incomplete. Considerando che lo sviluppo dei vaccini moderni è basato sui principi dell’immunità innata e acquisita è essenziale una sempre più completa conoscenza del sistema immunitario inteso come modulazione/regolazione molecolare della risposta infiammatoria e immunitaria in corso di tale infezione. Questo lavoro di tesi, suddiviso in tre diversi studi, ha l’intento di contribuire all’aumento delle informazioni riguardo l’interazione del sistema immunitario, con il virus della PRRS in condizioni di infezione naturale. L’obbiettivo del primo studio, intitolato “Associazione di cellule memoria, cellule citotossiche e cellule secernenti IFN- nella risposta immunitaria in corso di infezione naturale da Virus della Sindrome Riproduttiva e Respiratoria del Suino (PRRSV)” è stato di valutare l’attivazione e la modulazione della risposta immunitaria in suini naturalmente infetti da PRRSV rispetto ad un gruppo controllo non infetto. I parametri valutati sono stati la viremia mediante PCR, il titolo anticorpale mediante ELISA, il numero di cellule secernenti IFN- (IFN- SC) mediante tecnica ELISPOT e la fenotipizzazione di alcune sottopopolazioni linfocitarie (Cellule citotossiche, linfociti T memoria e linfociti T citotossici) mediante citofluorimetria a flusso. Dai risultati ottenuti è stato possibile osservare che l’attivazione della risposta immunitaria cellulo-mediata verso PRRSV appare ritardata durante l’infezione e che l’andamento, in termini di IFN- SC e dei cambiamenti delle sottopopolazioni linfocitarie, mostra comunque degli incrementi seppur successivi nel tempo. E’ stato inoltre osservato che gli andamenti delle diverse sottopopolazioni immunitarie cellulari appaiono temporalmente associati ai livelli di IFN- SC PRRSV-specifiche e ciò potrebbe essere interpretato sulla base del ruolo funzionale che tali sottopopolazioni linfocitarie potrebbero avere nella produzione/secrezione specifica della citochina immunoattivatrice IFN-. Questi dati inoltre supportano l’ipotesi che l’età degli animali alla comparsa dell’infezione o, come tipicamente ipotizzato nell’infezione da PRRSV, le differenti caratteristiche immunobiologiche dell’isolato di campo, sia condizioni critiche nell’ influenzare l’andamento qualitativo e quantitativo della risposta cellulo-mediata durante l’infezione naturale da PRRSV. Il secondo studio, dal titolo “Valutazione della risposta immunitaria nei confronti di una vaccinazione contro PCV2 in suini riscontrati PRRSV viremici e non viremici alla vaccinazione” ha avuto lo scopo di valutare se il virus della PRRS potesse andare ad interferire sull’attivazione della risposta immunitaria indotta da vaccinazione contro PCV2 nel suino. In questo lavoro sono stati arruolati 200 animali divisi in due gruppi, PCV2 Vaccinato (a 4 settimane di età) e PCV2 Non Vaccinato (controllo negativo). Alcuni suinetti di entrambi i gruppi, si sono naturalmente infettati con PRRSV, come determinato con l’analisi della viremia da PRRSV, per cui è stato possibile creare quattro sottogruppi, rispettivamente: PCV2 vaccinato - PRRSV viremico PCV2 vaccinato - PRRSV non viremico PCV2 non vaccinato - PRRSV viremico PCV2 non vaccinato - PRRSV non viremico Su questi quattro sottogruppi sono stati valutati i seguenti parametri: numero di cellule secernenti IFN- PCV2 specifiche, ed i titoli anticorpali mediante tecniche ELISA ed IPMA. Dall’analisi dei dati immunologici derivati dalle suddette tecniche è stato possibile dedurre che:  I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico già al periodo della vaccinazione (4 settimane di età) potrebbero essere messi in relazione ad una ridotta assunzione di colostro legata allo stato di viremia da PRRSV  Indipendentemente dallo stato viremico, i dati sierologici del gruppo vaccinato PCV2 provenienti sia da ELISA sia da IPMA non mostrano differenze statisticamente significative. Di conseguenza è possibile affermare che in questo caso PRRSV non interferisce con la risposta anticorpale promossa dal vaccino PCV2.  La risposta immunitaria cellulo-mediata, intesa come numero di cellule secernenti IFN- PCV2 specifiche nel gruppo PCV2 vaccinato PRRS viremico sembra essere compromessa, come viene infatti dimostrato dalla diminuzione del numero di cellule secernenti IFN- dopo la vaccinazione contro PCV2, comparata con il gruppo PCV2 vaccinato- non viremico. I dati evidenziano ed ulteriormente sostengono il ruolo inibitorio del virus della PRRSV sullo sviluppo ed attivazione della risposta immunitaria e come un infezione naturale ad età precoci possa influenzare negativamente la risposta immunitaria ad altri patogeni/antigeni. Il terzo studio, intitolato “Modulazione fenotipica di: monociti CD14+, cellule natural killer (NK), T natural killer (NKT) e sottopopolazioni linfocitarie T CD4+ e CD8+ durante stimolazione con killer peptide (KP) nella specie suina” ha avuto come scopo quello di stabilire se e come il Peptide Killer (KP) potesse modulare la risposta immunitaria in termini di attivazione di specifiche sottopopolazioni linfocitarie. Si tratta di un approccio preliminare anche ai fini di successivamente valutare tale KP in un potenziale ruolo antivirale o come adiuvante. In questo lavoro, periferal blood mononuclear cells (PBMC) suine sono state stimolate con KP a tre diverse concentrazioni (10, 20 e 40 g/ml) per tre diversi tempi (24, 48 e 72 ore). TEMPI DI STIMOLAZIONE (ore) CONCENTRAZIONE DI KP (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 Mediante la citometria a flusso è stato dunque possibile analizzare il comportamento qualitativo e quantitativo di alcune sottopopolazioni linfocitarie sotto lo stimolo del KP, tra cui: monociti, cellule Natural Killer (NK), cellule T Natural Killer (NKT) e linfociti T CD4 e CD8+. Dai dati ottenuti è stato possibile dedurre che: 1) KP promuove un’attivazione dei monociti dose-dipendente in particolare dopo 24 ore di stimolazione, inducendo uno “shift” fenotipico e di maturazione monocitaria maggiormente coinvolto nel sostegno della risposta innata/infiammatoria. 2) KP induce una forte modulazione dose-dipendente di cellule NK e NKT con un forte aumento della frazione delle cellule NKT rispetto alle NK, sottopopolazioni entrambe coinvolte nella citotossicità cellulare mediata da anticorpi (ADCC). L’aumento è riscontrabile soprattutto dopo 24 ore di stimolazione. 3) KP promuove una significativa attivazione della sottopopolazione del linfociti T citotossici (CTL). 4) Per quanto riguarda la marcatura CD4+/CD8+ è stato dimostrato che KP ha la capacità di modulare sia il fenotipo T helper che T citotossico, inducendo le cellule T helper ad acquisire CD8 diventando quindi doppio positive (CD4+CD8+) ed inducendo il fenotipo CTL (CD4-CD8+high) ad acquisire il fenotipo doppio positivo (CD4+CD8α+high). Molti dunque potrebbero essere gli effetti che il decapeptide KP potrebbe esercitare sulle diverse sottopopolazioni del sistema immunitario, per questo motivo va evidenziata la necessità di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna “abilità” di KP e che conducano successivamente alla scoperta del migliore utilizzo che si possa fare del decapeptide sia dal punto di vista vaccinale, terapeutico oppure sotto forma di adiuvante vaccinale.

Étude du rôle des régions variables 4 et 5 dans les changements de conformation de la gp120 du VIH-1

Le VIH infecte les cellules par fusion de sa membrane avec la membrane de la cellule cible. Cette fusion est effectuée par les glycoprotéines de l'enveloppe (Env) qui sont synthétisées en tant que précurseur, gp160, qui est ensuite clivé en gp120 et gp41. La protéine gp41 est la partie transmembranaire du complexe de l'enveloppe et l’ancre à la particule virale alors que la gp120 assure la liaison au récepteur cellulaire CD4 et corécepteur CCR5 ou CXCR4. Ces interactions successives induisent des changements de conformation d’Env qui alimentent le processus d'entrée du virus conduisant finalement à l'insertion du peptide de fusion de la gp41 dans la membrane de la cellule cible. La sous-unité extérieure gp120 contient cinq régions variables (V1 à V5), dont trois (V1, V2 et V3) étant capables d’empêcher l’adoption spontanée de la conformation liée à CD4. Cependant, le rôle de régions variables V4 et V5 vis-à-vis de ces changements de conformation reste inconnu. Pour étudier leur effet, des mutants de l'isolat primaire de clade B YU2, comprenant une délétion de la V5 ou une mutation au niveau de tous les sites potentiels de N-glycosylation de la V4 (PNGS), ont été générés. L'effet des mutations sur la conformation des glycoprotéines d'enveloppe a été analysé par immunoprécipitation et résonance de plasmon de surface avec des anticorps dont la liaison dépend de la conformation adopté par la gp120. Ni le retrait des PNGS de la V4 ni la délétion de V5 n’a affecté les changements conformationnels d’Env tels que mesurés par ces techniques, ce qui suggère que les régions variables V1, V2 et V3 sont les principaux acteurs dans la prévention de l’adoption de la conformation lié de CD4 d’Env.

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

Previous cancer vaccination trials often aimed to activate CD8(+) cytotoxic T-cell (CTL) responses with short (8-10mer) peptides and targeted CD4(+) helper T cells (TH) with HLA class II-binding longer peptides (12-16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8(+) T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4(+) TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4- and HLA-DR-restricted TH1 cells. Some short peptide-reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4(+) T-cell repertoire in many patients, facilitating a strong combined CD4(+)/CD8(+) T-cell response. Cancer Immunol Res; 4(1); 18-25. ©2015 AACR.

Short peptide alpha helices induced by multiple metal clips

Alpha helices are key structural components of proteins and important recognition motifs in biology. New techniques for stabilizing short peptide helices could be valuable for studying protein folding, modeling proteins, creating artificial proteins, and may aid the design of inhibitors or mimics of protein function. We previously reported* that 5-15 residue peptides, corresponding to the Zn-binding domain of thermolysin, react with [Pd(en)(ONO,),]in DMF-d’ and 90% H,O 10% DzO to form a 22-membered [Pd(en)(H*ELTH*)]2+ macrocycle that is helical in solution and acts as a template in nucleating helicity in both Cand N- terminal directions within the longer sequences in DMF. ~f~~&g7$$& d&qx~m ~. y AC&q& In water, however, there was less a-helicity observed, testifying to #..q,& &$--Lb &l-- &.$;,J~p?:~~q&~+~~ ’ w w the difficulty of fixing intramolecular amide NH...OC H-bonds in 6,“;;” ( k.$ U”C.a , p d$. competition with the H-bond donor solvent water. To expand the utility of [Pd(en)(H*XXXH*)]*+ as a helix- @r4”8 & oJ#:& &G& @-qd ,‘d@-gyp promoting module in solution, we now report the result that Ac- ‘$4: %$yyy + H*ELTH*H*VTDH*-NH,(l), AC-H*ELTH*AVTDYH*ELTH*- NH, (2) and AC-H*AAAH*H*ELTH*H*VTDH*-NH* (3) react with multiple equivalents of [Pd(en)(ONO,),] to produce exclusively 4-6 respectively in both DMF-d7 and water (90% Hz0 10% D,O). Mass spectrometry, 15N- and 2D ‘H- NMR spectroscopy, and CD spectra were used to characterise the structures 4-6, and their three dimensional structures were calculated from NOE restraints using simulated annealing protocols. Results demonstrate (a) selective coordination of metal ions at (i, i+4) histidine positions in water and DMF, (b) incorporation of 2 and 3 a turn-mimicking modules [Pd(en)(HELTH)]2+ in lo-15 residue peptides, and (c) facile conversion of unstructured peptides into 3- and 4- turn helices of macrocycles, with well defined a-helicity throughout and more structure in DMF than in water.

Designing supramolecular structures from models of cyclic peptide scaffolds with heterocyclic constraints

Cyclic peptides containing oxazole and thiazole heterocycles have been examined for their capacity to be used as scaffolds in larger, more complex, protein-like structures. Both the macrocyclic scaffolds and the supramolecular structures derived therefrom have been visualised by molecular modelling techniques. These molecules are too symmetrical to examine structurally by NMR spectroscopy. The cyclic hexapeptide ([Aaa-Thz](3), [Aaa-Oxz](3)) and cyclic octapeptide ([Aaa-Thz](4), [Aaa-Oxz](4)) analogues are composed of dipeptide surrogates (Aaa: amino acid, Thz: thiazole, Oxz: oxazole) derived from intramolecular condensation of cysteine or serine/threonine side chains in dipeptides like Aaa-Cys, Aaa-Ser and Aaa-Thr. The five-membered heterocyclic rings, like thiazole, oxazole and reduced analogues like thiazoline, thiazolidine and oxazoline have profound influences on the structures and bioactivities of cyclic peptides derived therefrom. This work suggests that such constrained cyclic peptides can be used as scaffolds to create a range of novel protein-like supramolecular structures (e.g. cylinders, troughs, cones, multi-loop structures, helix bundles) that are comparable in size, shape and composition to bioactive surfaces of proteins. They may therefore represent interesting starting points for the design of novel artificial proteins and artificial enzymes. (C) 2002 Elsevier Science Inc. All rights reserved.

Cosegregation analysis of natriuretic peptide genes and blood pressure in the spontaneously hypertensive rat

1. The natriuretic peptide precursor A (Nppa) and B (Nppb) genes are candidate genes for hypertension and cardiac hypertrophy in the spontaneously hypertensive rat (SHR). The purpose of the present study was to determine the role of the Nppa and Nppb genes in the development of hypertension in the SHR. 2. A cohort (n = 162) of F2 segregating intercross animals was established between strains of hypertensive SHR and normotensive Wistar-Kyoto rats. Blood pressure and heart weight were measured in each rat at 12-16 weeks of age. Rats were genotyped using 11 informative microsatellite markers, distributed in the vicinity of the Nppa marker on rat chromosome 5 including an Nppb marker. The phenotype values were compared with genotype using the computer package MAP-MAKER 3.0 (Whitehead Institute, Boston, MA, USA) to determine whether there was a link between the genetic variants of the natriuretic peptide family and blood pressure or cardiac hypertrophy. 3. A strong correlation was observed between the Nppa marker and blood pressure. A quantitative trait locus (QTL) for blood pressure on chromosome 5 was identified between the Nppa locus and the D5Mgh15 marker, less than 2 cM from the Nppa locus. The linkage score for the blood pressure QTL on chromosome 5 was 3.8 and the QTL accounted for 43% of the total variance of systolic blood pressure, 54% of diastolic blood pressure and 59% of mean blood pressure. No association was found between the Nppb gene and blood pressure. This is the first report of linkage between the Nppa marker and blood pressure in the rat. There was no correlation between the Nppa or Nppb genes or other markers in this region and either heart weight or left ventricular weight in F2 rats. 4. These findings suggest the existence of a blood pressure-dependent Nppa marker variant or a gene close to Nppa predisposing to spontaneous hypertension in the rat. It provides a strong foundation for further detailed genetic studies in congenic strains, which may help to narrow down the location of this gene and lead to positional cloning.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.