A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LTG33D), originally produced by some enterotoxigenic Escherichia coil (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LIG33D. Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LTG33D elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LTG33D. Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LTG33D. Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 mu g of rhBMP-2, (3) laser and 7 mu g of rhBMP-2, (4) 7 mu g of rhBMP-2/monoolein gel, (5) laser and 7 mu g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-beta-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 degrees C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan beta-1,3 or beta-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in beta-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 degrees C and 81.3 degrees C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 degrees C. the enzymatic assays demonstrated that XegA is more active in its monomeric state. (c) 2012 Elsevier B.V. All rights reserved.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Isolation and identification of molecular partners of the proteins encoded by the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs

Ziel dieser Arbeit war es, die funktionelle Bedeutung des Drosophila melanogaster tumor suppressor Gens lethal(2)tumorous imaginal discs (l(2)tid) durch die Identifikation von molekularen Partnern der vom Gen kodierten Proteine zu etablieren. Mit dem Screening einer Expressionsbibliothek mittels des Hefe-Di-Hybrid-Systems wurde das Protein Patched (Ptc) als ein neues Tid-bindendes Protein identifiziert. Ptc ist ein Zentralregulator der Hedhehog-Signalkette. Diese ist in der Entwicklung konserviert und in manchen humanen Krebsarten verwickelt. Die Tid/Ptc-Interaktion wurde mittels unabhängigen biochemischen Methoden wie dem GST-pulldown-Test oder der Immunopräzipitation überprüft. Außerdem ergaben funktionelle Studien in tumorosen Imaginalscheiben einen möglichen inhibitorischen Effekt von Tid über die Hh Signaltransduktion.Im letzten Teil dieser Arbeit wurde die Interaktion zwischen Tid und dem E-APC-Protein (Adenomatous polyposis coli) bewiesen. Polakis und seine Gruppe zeigten durch Studien mit dem Hefe-Di-Hybrid-System und in vitro, dass das hTid mit dem APC-Protein interagiert. Um dies auch auf Drosophila-Ebene zu überprüfen, wurden Immunopräzipitation-Studien mit den Drosophila-Gegenstücken durchgeführt. Diese Studien zeigen zum ersten Mal eine direkte Interaktion beider Proteine in vivo.

Pigmentbindung verschiedener Mitglieder der erweiterten Chlorophyll a/b-Proteinfamilie und des wasserlöslichen Chlorophyll-Proteins WSCP

In dieser Arbeit wurde die Pigmentbindung verschiedener Pflanzenproteine untersucht, um daraus Rückschlüsse auf ihre Funktion zu ziehen. PsbS, die S-Untereinheit des Photosystems II, konnte mit Pigmenten isoliert werden. Es wurde kein Hinweis auf eine spezifische Wechselwirkung der Chromophore gefunden, Ergebnisse wie pigmentabhängig stärkere Helixbildung unterstützen jedoch die Vermutung, PsbS fungiere als transienter Pigmentcarrier. Die Sequenzverwandten OHP, Sep1 und Sep2 binden entweder keine Pigmente oder nur so schwach, dass eine Bindung mit den verwendeten Methoden nicht nachweisbar ist.WSCP aus Blumenkohl ist ein wasserlösliches chlorophyllbindendes Protein mit unbekannter Funktion. In dieser Arbeit wurde ein rekombinantes WSCP mit N-terminal angehängtem His-Tag hergestellt und überexprimiert. WSCP-his tetramerisiert pigmentabhängig und bindet Chlorophylle, nicht aber Carotinoide. In seinen biochemischen und spektroskopischen Eigenschaften gleicht das rekombinante dem nativen WSCP und kann als Werkzeug für Untersuchungen zur Funktion herangezogen werden. Rekonstitutionsexperimente mit Chlorophyll-Derivaten zeigten, dass der Phytolrest für die Oligomerisierung des Proteins verantwortlich ist. WSCP bindet außerdem die Chlorophyll-Vorstufen Chlorophyllid und Mg-Protoporphyrin IX. Es könnte sich um ein Carrierprotein handeln, welches die Vorstufen von der Chloroplastenhülle durch das Stroma zur Thylakoidmembran transportiert. Der Fall eines chlorophyllbindenden Pflanzenproteins ohne Carotinoide ist einmalig. Messungen zu Photostabilität und Singulettsauerstoffbildung zeigten, dass es dennoch gebundenes Chlorophyll vor photooxidativer Schädigung schützt.

Untersuchungen zur Rolle des BH3-only-Proteins Bid bei der Stress-induzierten Apoptose in Karzinomzelllinien

Als BH3-only Protein gehört Bid zu den proapoptotischen Mitgliedern der Bcl-2 Familie, die während der Apoptose die Freisetzung Caspase-aktivierender Proteine aus den Mitochondrien kontrollieren. Bid zählt zu den potentesten BH3-only Proteinen und wird von vielen transformierten und nichttransformierten Zellen konstitutiv exprimiert. Ziel dieser Arbeit war es, Bid durch RNA-Interferenz stabil zu depletieren, um Bid-abhängige Apoptosewege in HeLa Zervixkarzinomzellen zu identifizieren, die von intrinsischen Stressstimuli sowie von konventionellen und neuartigen Chemotherapeutika induziert werden. Da Bid im Todesrezeptor-vermittelten Signalweg der Apoptose durch Caspase-8 gespalten und aktiviert wird, waren die Bid-depletierten Zellen signifikant vor der Fas/CD95-, TRAIL- oder TNF-α-induzierten Apoptose geschützt und zeigten nach Exposition mit allen drei Todesrezeptorliganden eine drastisch reduzierte Effektorcaspase-Aktivität und eine höhere Proliferationsrate als die Kontrollzellen. Eine ektopische Bidexpression in Bid knock down (kd) Zellen hob die Protektion vor der Fas- und TRAIL-induzierten Apoptose auf. Der Proteasominhibitor Epoxomicin, der Proteinkinase-Inhibitor Staurosporin oder die ER Stress-induzierenden Agenzien Tunicamycin, Thapsigargin und Brefeldin A lösten hingegen einen Bid-unabhängigen Zelltod aus. Allerdings konnten subletale Tunicamycin- oder Thapsigarginkonzentrationen HeLa Zellen für die TRAIL-induzierte Apoptose sensitivieren. Da der Synergieeffekt auf einer ER Stress-vermittelten Amplifizierung des Todesrezeptorwegs beruhte, zu der eine Tunicamycin-induzierte Steigerung der Expression des Todesrezeptors DR5 signifikant beitrug, erfolgte diese Sensitivierung nur in Bid-profizienten Zellen. Bid war in HeLa Zellen außerdem an der apoptotischen Signalkaskade beteiligt, die von den DNA-schädigenden Agenzien Etoposid, Doxorubicin und Oxaliplatin (Oxa) ausgelöst wird. Nach Behandlung mit Oxa zeigten die Bid kd Zellen eine verzögerte Caspase-2, -3, -8 und -9 Aktivierung, einen geringeren Verlust des mitochondrialen Membranpotentials sowie eine reduzierte Apoptose- und eine höhere Proliferationsrate als Bid-profiziente Zellen. Neben Bid war ein weiteres BH3-only Protein, Puma, an der Oxa-induzierten Effektorcaspase-Aktivierung beteiligt, da eine Puma-spezifische siRNA unabhängig vom Bidstatus der Zellen antiapoptotisch wirkte. Im letzten Teil der Arbeit wurde untersucht, welche Proteasen für die durch gentoxische Agenzien induzierte Spaltung und Aktivierung von Bid verantwortlich sind. Obwohl Caspasen für die Exekutionphase der Oxa-induzierten Apoptose notwendig waren, trugen sie weder zur initialen Bidaktivierung noch zur mitochondrialen Depolarisierung bei, da sie erst postmitochondrial aktiviert wurden. Konventionelle Calpaine hingegen wurden nach DNA-Schädigung bereits stromaufwärts der Mitochondrien aktiviert und der Calpaininhibitor Calpeptin reduzierte nicht nur die Bid- und Caspasespaltung, sondern auch die mitochondriale Depolarisierung signifikant. Diese Protektion durch Calpeptin fiel in Bid-depletierten Zellen signifikant geringer als in Bid-profizienten Kontrollzellen aus. Auch war in Oxa-behandelten Bid kd Zellen, die eine durch Caspase-2, -3 und -8 nicht spaltbare Bidmutante exprimierten, trunkiertes Bid nachweisbar, dessen Generierung durch Calpain-, aber nicht durch Caspaseinhibierung verhindert werden konnte. Zusammenfassend deuten diese Ergebnisse auf eine Calpain-abhängige Bidaktivierung stromaufwärts der Mitochondrien hin und zeigen, dass die BH3-only Proteine Bid und Puma wichtige Vermittler der Oxa-induzierten Apoptose in HeLa Zellen darstellen.

Functional characterization of modified vaccinia virus Ankara-encoded anti-apoptotic proteins

Candidate vaccines based on the highly attenuated orthopoxvirus strain MVA are tested against various infectious and cancer diseases and, more profound, vaccines based on wildtype and recombinant viruses have been found safe and immunogenic in clinical trials. Compared to conventional vaccine strains, MVA lacks many functional genes for potentially important regulators of virus-host interactions. However, some gene functions responsible for counteraction of cellular antiviral pathways are still conserved in the genome of MVA and the inhibition of apoptosis seems to be one important mechanism, the virus is still able to interact with.rnrnVaccinia viruses encode several proteins which prevent the induction of virus-induced apoptosis. The vaccinia virus anti-apoptotic protein F1 was shown to counteract the activation of the mitochondrial pathway of apoptosis in a highly effective manner. Another vaccinia virus protein, N1, like F1 shows structural and functional similarity to members of the cellular anti-apoptotic bcl-2 family and was also shown to inhibit apoptosis. The vaccinia virus early protein E3 inhibits programmed cell death by binding to and sequestration of dsRNA molecules, normally inducing cellular antiviral pathways also driving the induction of apoptosis. All three anti-apoptotic genes were functionally analyzed during this work.rn

Conjugation of Toll Like Receptor 7 agonist through conjugation to immunogenic proteins. Synthesis, characterization, formulation and pre-clinical evaluation

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates