Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (−/−) cells, and analyzed cell–cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.

Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands

Recent data suggest that survival of resting, naïve T cells requires an interaction with self MHC molecules. From analysis of the class I MHC-restricted T cell receptor transgenic strain OT-I, we report a different response. Rather than merely surviving, these T cells proliferated slowly after transfer into T-depleted syngeneic hosts. This expansion required both T cell “space” and expression of normal levels of self class I MHC molecules. Furthermore, we demonstrate that during homeostatic expansion in a suitable environment, naïve phenotype (CD44low) OT-I T cells converted to memory phenotype (CD44med/high), despite the absence of foreign antigenic stimulation. On the other hand, cells undergoing homeostatic expansion did not acquire cytolytic effector function. The significance of these data for reactivity of T cells with self peptide/MHC ligands and the implications for normal and abnormal T cell homeostasis are discussed.

Oryza sativa PSK gene encodes a precursor of phytosulfokine-α, a sulfated peptide growth factor found in plants

Phytosulfokine-α [PSK-α, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-α precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. [Ser4]PSK-α was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-α molecules have never been identified in any intact plant, reverse transcription–PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-α may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.

Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: Reagents for tumor immunotherapy

Studies in melanoma patients have revealed that self proteins can function as targets for tumor-reactive cytotoxic T lymphocytes (CTL). One group of self proteins MAGE, BAGE, and GAGE are normally only expressed in testis and placenta, whilst another group of CTL recognized proteins are melanocyte-specific differentiation antigens. In this study we have investigated whether CTL can be raised against a ubiquitously expressed self protein, mdm-2, which is frequently overexpressed in tumors. The observation that T-cell tolerance is self major histocompatibility complex-restricted was exploited to generate CTL specific for an mdm-2 derived peptide presented by nonself major histocompatibility complex class I molecules. Thus, the allo-restricted T-cell repertoire of H-2d mice was used to isolate CTL specific for the mdm100 peptide presented by allogeneic H-2Kb class I molecules. In vitro, these CTL discriminated between transformed and normal cells, killing specifically Kb-positive melanoma and lymphoma tumors but not Kb-expressing dendritic cells. In vivo, the CTL showed antitumor activity and delayed the growth of melanoma as well as lymphoma tumors in H-2b recipient mice. These experiments show that it is possible to circumvent T-cell tolerance to ubiquitously expressed self antigens, and to target CTL responses against tumors expressing elevated levels of structurally unaltered proteins.

Targeting cyclin-dependent kinases in Drosophila with peptide aptamers

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.

An artificial cell-cycle inhibitor isolated from a combinatorial library

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21Cip1, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

In vivo antagonism of a T cell response by an endogenously expressed ligand

3.L2 T cell receptor transgenic T cells are activated by the 64–76 peptide of the mouse hemoglobin d β chain [Hb(64–76)], and their response is antagonized by the position 72 alanine substitution of this peptide (A72). To test the effect of this altered peptide ligand (APL) on 3.L2 T cell function in vivo, a transgene expressing A72 in major histocompatibility complex II positive cells (A72tg) has been introduced into mice. We demonstrate that 3.L2 T cells, when transferred to A72tg+ mice show a dramatically reduced proliferative response to Hb(64–76). Identical decreased responses were observed using T cells that developed in either A72tg+ or A72tg− hosts. This affect was not attributable to diminished precursor frequency, anergy, or competition for binding to I-Ek molecules. These results unequivocally demonstrate in vivo antagonism by an endogenous APL and characterize a class of self-peptides that, although inefficient in causing deletion in the thymus, effectively modulate T cell responses in the periphery.

Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. METPRC mutations result in increased kinase activity and—in some instances, i.e., M1250T substitution—in changes in substrate specificity. A direct correlation occurs between the transforming potential of METPRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y1349VHVNATY1356VNV) located in the receptor tail. Substitution of these “docking tyrosines” with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (YpVNV) inhibits invasive growth induced by METPRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene.

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2′-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.