Peptide-binding Domain Research Articles

113. HSV Vector Development for Targeted Gene Delivery

Gene therapy often requires selective gene delivery to specific cellular subpopulations to provide the most effective therapeutic outcome and avoid off-target effects. This can be accomplished by viral vector targeting involving replacement of the natural virus-cell interactions that trigger virus entry, with novel cell-specific interactions, thereby permitting delivery of therapeutic transgenes to specific cell types. Strategies for full retargeting of HSV require (i) mutagenesis-mediated virus detargeting from its cognate receptors (HVEM and nectin1) recognized by the virus attachment/entry component glycoprotein D (gD) and (ii) the introduction of new ligands into gD that allow entry through recognition of cognate cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptors GFRa1 or TrkA, we employed the ligands GDNF and NGF, respectively. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRa1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient cells in a GFRa1-dependent manner. Likewise, TrkA-dependent virus entry was observed using ppNGF as a targeting ligand. Although propagation of completely retargeted viruses (mutationally inactivated for both HVEM and nectin1 binding) can be achieved in complementing cell lines that express the target receptor, we observed differences among cell lines in retargeted virus spread. HSV receptor-deficient J1.1-2 and B78H1 cells engineered to express the target receptors allowed virus entry, but only B78H1 based cells displayed observable spread. U2OS cells engineered to express GFRa1 demonstrated the most robust virus entry and spread, but these cells express abundant nectin1, providing strong selective pressure for reversion of the nectin1 binding defect of fully retargeted viruses during propagation. In one approach to overcome this problem, we developed genetic selection methods to isolate retargeted virus variants that both entered and spread on J1.1-2 cells transduced with the target receptor. We found that selected variants had acquired mutations in other envelope glycoproteins, including one glycoprotein involved in envelope-cell fusion events (gH) and two that were previously shown to contribute to the spreading process (gE and gI). We expect that the combination of virus mutants and engineered cell lines for efficient production of stably retargeted HSV gene therapy vectors will allow us to achieve HSV retargeting to a broader range of receptors.

Abstract 659: Inhibition of vWF Secretion with Novel G alpha12 N-terminal alpha-SNAP Binding Domain Peptide Increases Survival in Septic Rodents

Severe sepsis is associated with disseminated intravascular coagulation (DIC) as a result of interdependent mechanisms of systemic intravascular inflammation, microvascular thrombosis, and thrombocytopenia. Currently, no drug is available to concomitantly treat these events in sepsis. A poorly understood mechanism and yet critical determinant of sepsis-induced microvascular thrombosis is von Willebrand Factor (vWF) secretion by activated endothelial cells. We recently discovered that heterotrimeric G protein alpha subunit G alpha 12 plays a critical role in basal and evoked vWF secretion by endothelial cells by promoting Weibel-Palade body (WPB) exocytosis. Based on the observed interaction of G alpha 12 with alpaSNAP, a critical member of the exocyst complex required for plasma membrane fusion and exocytosis of WPB contents, we generated a myristoylated Galpha12 N-terminal alphaSNAP Binding Domain (Myr-SBD) blocking peptide and tested the hypothesis that this would selectively and potently inhibit Galpha12 interaction with alphaSNAP and thereby block vWF secretion, limit platelet adhesion and prevent microvascular thrombosis associated with cecal ligation and puncture (CLP) induced sepsis. CLP-induced fulminant sepsis in rats and mice was associated with a 2-3-fold increase in plasma vWF within first 24 hrs. Importantly, we observed reduced plasma vWF levels 24 hrs after CLP surgery in mice given a one-time i.v. bolus (2 μmol/kg) of micellar Myr-SBD at the time of surgery as compared to Myr-scrambled peptide or vehicle only group. Strikingly, this was associated with increased survival without adversely inducing hemorrhage and vascular leakage. Furthermore, and consistent with the hypothesis that Gα12-dependent increase in vWF secretion during sepsis leads to poor outcome, control WT mice succumbed to sepsis in less than 96 hrs whereas 80% of Gα12-/- mice shown previously to have significantly reduced plasma vWF levels survived. Inhibition of Galpha12/alphaSNAP dependent vWF secretion may therefore be an effective strategy for blocking microvascular thrombosis, disseminated intravascular coagulation, and death due to sepsis.

Highly clonal regulatory T-cell population in follicular lymphoma – inverse correlation with the diversity of CD8+ T cells

The immune microenvironment in follicular lymphoma (FL) plays an important role in controlling disease characteristics. To characterize the T-cell receptor (TCR) repertoire in follicular lymphoma (FL) tissues, we applied a next-generation sequencing platform and deeply sequenced TCR cDNAs of T-cell subset populations present in pretreatment FL biopsy specimens. T regulatory cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who had not received anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both human leukocyte antigen (HLA) class I and II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T-cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease.

The Interaction of Hsp70 with Phosphatidylserine Membranes is Mediated by a Highly Positive Region of the Molecule

Heat shock proteins (hsp) participate in many cellular processes during normal physiological conditions. In particular, they are involved in protein folding and are referred to as molecular chaperones. During stress conditions, hsp participate in the repair and recovery from an insult and confer protection from subsequent stresses. In addition, they are released into the extracellular milieu where they act as signaling molecules directed at activating the immune system to avoid the propagation of the insult. Hsp70, the major inducible form of the hsp family, does not contain any consensus secretory signal that predicts its export via the classical ER-Golgi secretory pathway. The proposed mechanism for the release of Hsp70 requires an initiated translocation into the plasma membrane. However, Hsp70 does not contain any hydrophobic domain that can predict its insertion into lipid membranes. Using an in vitro liposome insertion assay, we have determined that Hsp70 displays selectivity for negatively charge lipids and its insertion is enhanced by a decrease in the membrane fluidity. The region that is inserted into the lipid membrane has been mapped toward the C-terminus end, which contains the peptide binding domain. Lipid recognition/insertion requires a highly positive region on the molecule. Finally, the addition of ADP, but not ATP, reduced membrane insertion, suggesting that the interaction with the lipid bilayer is dependent on the conformation of the molecule.Supported by NIH R01 GM098455.

Combinatorial screening for specific drug solubilizers with switchable release profiles.

Polymer-block-peptide conjugates are tailored to render hydrophobic small molecule drugs water soluble. The combinatorial strategy selects for bioconjugates that exhibit sequence-specific solubilization and switchable release profiles of the cargo through incorporation of a disulfide linker moiety into the peptide-library design. While the study focused on the photosensitizer m-THPC and reductive carrier cleavage, the approach is generic and might be expanded toward a broad range of poorly soluble small-molecule drugs and other selective cleavage mechanisms to disassemble a peptide binding domain of the bioconjugate-based solubilizer.

Pepper heat shock protein 70a interacts with the type III effector AvrBsT and triggers plant cell death and immunity.

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.

Chronic inflammation and angiogenic signaling axis impairs differentiation of dental-pulp stem cells.

Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.

The RapP-PhrP quorum-sensing system of Bacillus subtilis strain NCIB3610 affects biofilm formation through multiple targets, due to an atypical signal-insensitive allele of RapP.

The genome of Bacillus subtilis 168 encodes eight rap-phr quorum-sensing pairs. Rap proteins of all characterized Rap-Phr pairs inhibit the function of one or several important response regulators: ComA, Spo0F, or DegU. This inhibition is relieved upon binding of the peptide encoded by the cognate phr gene. Bacillus subtilis strain NCIB3610, the biofilm-proficient ancestor of strain 168, encodes, in addition, the rapP-phrP pair on the plasmid pBS32. RapP was shown to dephosphorylate Spo0F and to regulate biofilm formation, but unlike other Rap-Phr pairs, RapP does not interact with PhrP. In this work we extend the analysis of the RapP pathway by reexamining its transcriptional regulation, its effect on downstream targets, and its interaction with PhrP. At the transcriptional level, we show that rapP and phrP regulation is similar to that of other rap-phr pairs. We further find that RapP has an Spo0F-independent negative effect on biofilm-related genes, which is mediated by the response regulator ComA. Finally, we find that the insensitivity of RapP to PhrP is due to a substitution of a highly conserved residue in the peptide binding domain of the rapP allele of strain NCIB3610. Reversing this substitution to the consensus amino acid restores the PhrP dependence of RapP activity and eliminates the effects of the rapP-phrP locus on ComA activity and biofilm formation. Taken together, our results suggest that RapP strongly represses biofilm formation through multiple targets and that PhrP does not counteract RapP due to a rare mutation in rapP.

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptide-binding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples >10 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science.

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.

The anti-inflammatory role of nuclear factor-kappa B essential modulator binding domain peptide in a rabbit osteomyelitis model

Objective To investigate the anti-inflammatory role of nuclear factor-kappa B essential modulator binding domain (NBD) peptide in a rabbit osteomyelitis model.Methods Twenty-four male rabbits were used to establish models of chronic osteomyelitis with Staphylococcus aureus (ATCC25923).The animals weighed from 2 to 3 kilograms (average,2.5 kg).Three random rabbits were sacrificed 3 weeks later to verify successful modeling of chronic osteomyelitis by radiological,bacteriological and pathological observations.The remaining 21 successful models were randomized into 3 groups(n =7).The blank group received no further treatment,the control group received debridement,and the experimental group received debridement plus NBD peptide (3 mg/kg body weight).C reactive protein(CRP),erythrocyte sedimentation rate (ESR),leucocyte count,and HE staining of focus tissue were observed l to 6 weeks after intervention.Results At pre-intervention,and 1 to 6 weeks post-intervention,the CRP and ESR levels showed a decreasing trend in all the 3 groups while the leucocyte count showed an increasing trend in the blank and control groups but a decreasing trend in the experimental group.At one week post-intervention,there was a significant difference in CRP between the 3 groups (P ＜ 0.05).At 2 weeks post-intervention,the CRP level in the experimental group was significantly different from that in the blank or control group (P ＜ 0.05).At 1 to 6 weeks post-intervention,there was a significant difference in the leucocyte count between the 3 groups (P ＜ 0.05).At 6 weeks post-intervention,HE staining showed even bony staining,no obvious formation of dead bone,dense fibroplasias in the marrow cavity,and no obvious inflammatory cells in the experimental group.Conclusion NBD peptide can block inflammation in a rabbit model of tibia osteomyelitis. Key words: Peptides; Transcription factors; Osteomyelitis

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

Characterization and expression analysis of a mitochondrial heat-shock protein 70 gene from the Antarctic moss Pohlia nutans

Under stress conditions, the mitochondrial respiratory chain generates reactive oxygen species that cause oxidative damage to lipids, proteins and nucleic acids. Mitochondrial chaperones are essential for repairing denatured proteins in plants confronted with diverse stresses. However, their functions have not been well characterized in lower plants such as bryophytes. Here, we cloned a heat-shock 70 protein gene (designated PnHSP70) from the Antarctic moss Pohlia nutans and investigated its transcription levels under stress conditions. The PnHSP70 cDNA encoded a polypeptide of 678 amino acids with an ATPase domain, a substrate peptide-binding domain and a C-terminal domain. Sequence alignment and phylogenetic analysis showed that PnHSP70 was homologous to other known mitochondrial HSP70 proteins. In addition, subcellular localization analysis in which the transient expression of PnHSP70-green fluorescent protein was observed in Arabidopsis thaliana mesophyll protoplasts revealed that PnHSP70 was targeted to mitochondria. Real-time PCR analysis showed that low or high temperature, salinity, drought or PEG treatments as well as UV-B radiation could induce up-regulation of PnHSP70 expression levels. The plant hormones abscisic acid and jasmonic acid also up-regulated the expression levels of PnHSP70. Together, our results revealed that PnHSP70, as a mitochondrial chaperone, might play an important role in the adaptation of the Antarctic moss P. nutans to the polar environment.

Molecular cloning and characterization of an Hsp70 gene from the bloom green alga Chaetomorpha valida (Cladophorales, Chlorophyta)

The full-length complimentary DNA (cDNA) of an Hsp70 gene from the bloom forming green alga Chaetomorpha valida (designated as CvHsp70) was isolated based on homologous cloning and the rapid amplification of cDNA ends (RACE) (Accession: KF812520). It was 2,225 bp, with an open reading frame of 1,995 bp. The deduced 664 amino acids possessed an ATPase domain of 429 amino acids, a substrate peptide-binding domain of 124 amino acids and a C-terminus domain of 111 amino acids. About 46–71 % similarities were detected between CvHsp70 and other 39 known Hsp70s of algae and higher plants; highly conserved motifs were also indentified in CvHsp70, which suggest the conservation of Hsp70 function. Although CvHsp70 presented the endoplasmic reticulum (ER) consensus sequence HDEL in the C-terminal, the subcellular prediction programs presumed that it was targeted to cytoplasm or ER; In the phylogenetic trees, the CvHsp70 formed a basal clade in the cytosolic group with high node support, which implies that it is possibly a chimera of ER and cytosolic Hsp70. Real-time PCR detection revealed the CvHsp70 expression was dramatically upregulated by temperature, desiccation, and salinities induction, which suggests that CvHsp70 might act as a stress responsive gene involved with protecting C. valida from abiotic stresses, thus indirectly facilitating growth and accumulation in unfavorable natural environments. In vitro expression showed one distinct band existed at ~72 kDa, and western blot detection showed that it was positive to the anti-His antibody with high specificity. This is the first study to characterize Hsp70 gene from Cladophoraceae species. Our results facilitate deeper analysis Hsp70 detailed functions in abiotic stress tolerance of C. valida and shed more light on the intrinsic mechanisms underlying the seasonal abundance.

An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage.We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.

Exogenously delivered heat shock protein 70 displaces its endogenous analogue and sensitizes cancer cells to lymphocytes-mediated cytotoxicity.

Hsp70 chaperone is known to stimulate anti-tumour immunity in a variety of cancer models. Here we demonstrated that the addition of purified recombinant Hsp70 to the culture medium facilitated cancer cell cytolysis by lymphocytes. Importantly, exogenous Hsp70 triggered secretion of the intracellular Hsp70 to a cell surface and extracellular milieu, which played a role in cytolysis because down-regulation of the endogenous Hsp70 reduced both its presence at the cell surface and the lymphocyte-mediated cytolysis. Inhibitors that target both the ATPase and the peptide-binding domains of Hsp70 molecule potently decreased its anti-tumor effect. Using a variety of cell transport markers and inhibitors, we showed that the exchange of exogenous and intracellular Hsp70 is supported by classical and non-classical transport pathways, with a particular role of lipid rafts in the chaperone's intracellular transport. In conclusion, exogenous Hsp70 can eject endogenous Hsp70, thus exerting anticancer activity.

Two Polymorphisms Facilitate Differences in Plasticity between Two Chicken Major Histocompatibility Complex Class I Proteins

Major histocompatibility complex class I molecules (MHC I) present peptides to cytotoxic T-cells at the surface of almost all nucleated cells. The function of MHC I molecules is to select high affinity peptides from a large intracellular pool and they are assisted in this process by co-factor molecules, notably tapasin. In contrast to mammals, MHC homozygous chickens express a single MHC I gene locus, termed BF2, which is hypothesised to have co-evolved with the highly polymorphic tapasin within stable haplotypes. The BF2 molecules of the B15 and B19 haplotypes have recently been shown to differ in their interactions with tapasin and in their peptide selection properties. This study investigated whether these observations might be explained by differences in the protein plasticity that is encoded into the MHC I structure by primary sequence polymorphisms. Furthermore, we aimed to demonstrate the utility of a complimentary modelling approach to the understanding of complex experimental data. Combining mechanistic molecular dynamics simulations and the primary sequence based technique of statistical coupling analysis, we show how two of the eight polymorphisms between BF2*15∶01 and BF2*19∶01 facilitate differences in plasticity. We show that BF2*15∶01 is intrinsically more plastic than BF2*19∶01, exploring more conformations in the absence of peptide. We identify a protein sector of contiguous residues connecting the membrane bound α3 domain and the heavy chain peptide binding site. This sector contains two of the eight polymorphic residues. One is residue 22 in the peptide binding domain and the other 220 is in the α3 domain, a putative tapasin binding site. These observations are in correspondence with the experimentally observed functional differences of these molecules and suggest a mechanism for how modulation of MHC I plasticity by tapasin catalyses peptide selection allosterically.

Six-cell penetrating peptide-based fusion proteins for siRNA delivery

The siRNA deliveries, for the siRNA’s high negative charge, short serum half life, poor cellular internalization, etc, are still the key barriers for its application in clinic. In this study, several cell penetrating peptide (CPP) and dsRNA binding domain (dsRBD)-based fusion proteins have been developed and screened as the siRNA vector. The siRNA binding ability was measured by the agarose gel retardation, the cell uptaking was characterized under fluorescence microscopy, and further more RNAi effect was evaluated on the endogenous (GAPDH, western blot) and exogenous (GFP, flow cytometry analysis) genes in HeLa cell. Finally, the cytotoxicity was assessed on HeLa cells using cell counting kit-8. The efficiency of siRNA delivery by the CPP-dsRBD fusion protein was the CPP and the dsRBD dependent. Three fusion proteins showed similar efficiency of siRNA delivery when comparing to Lipofectamine RNAi Max as the siRNA carrier. These results indicated that these CPP-dsRBD-based fusion proteins were promising candidates as siRNA carriers.