Discrete Peptides Research Articles

Parathyroid Hormone Related Protein (PTHrP)-Associated Molecular Signatures in Tissue Differentiation and Non-Tumoral Diseases.

Parathyroid-hormone-related protein (PTHrP) is encoded by the PTHLH gene which, via alternative promoter usage and splicing mechanisms, can give rise to at least three isoforms of 139, 141, and 173 amino acids with distinct C-terminals. PTHrP is subjected to different post-translational processing that generates smaller bioactive forms, comprising amino terminus, mid-region (containing a nuclear/nucleolar targeting signal), and carboxy terminus peptides. Both the full-length protein and the discrete peptides are key controllers of viability, proliferation, differentiation, and apoptosis in diverse normal and pathological biological systems via the reprogramming of gene expression and remodulation of PKA or PKC-mediated signalization mechanisms. The aim of this review is to pick up selected studies on PTHrP-associated signatures as revealed by molecular profiling assays, focusing on the available data about exemplary differentiating, differentiated, or nontumoral cell and tissue models. In particular, the data presented relate to adipose, bone, dental, cartilaginous, and skin tissues, as well as intestinal, renal, hepatic, pulmonary, and pancreatic epithelia, with a focus on hepatic fibrosis-, pancreatitis-, and diabetes-related changes as diseased states. When reported, the biochemical and/or physiological aspects associated with the specific molecular modulation of gene expression and signal transduction pathways in the target model systems under examination are also briefly described.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

Parathyroid hormone-related protein (PTHrP)-dependent modulation of gene expression signatures in cancer cells.

PTHrP is encoded by PTHLH gene which can generate by alternative promoter usage and splicing mechanisms at least three mature peptides of 139, 141 and 173 amino acids with distinct carboxy terminus. PTHrP may undergo proteolytic processing into smaller bioactive forms, comprising an amino terminus peptide, which is the mediator of the "classical" PTH-like effect, as well as midregion and carboxy terminus peptides that act as multifaceted critical regulator of proliferation, differentiation and apoptosis via the reprogramming of gene expression in normal and neoplastic cells. Moreover, a nuclear/nucleolar localization signal sequence is present in the [87-107] domain allowing PTHrP nuclear import and "intracrine" effect additional to the autocrine/paracrine one. Within the large number of data available in the literature on PTHrP bioactivities, the goal of this chapter is to pick up selected studies that report the detection of molecular signatures of cancer cell exposure to PTHrP, either as full-length protein or discrete peptides, demonstrated by individual gene or whole genome expression profiling, briefly recapitulating the biological implications associated with the specific gene activation or silencing.

A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 μm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

Deciphering CD4+ T cell specificity using novel MHC-TCR chimeric receptors.

αβ T cell antigen receptors (TCRs) bind complexes of peptide and major histocompatibility complex (pMHC) with low affinity, which poses a considerable challenge for the direct identification of αβ T cell cognate peptides. Here we describe a platform for the discovery of MHC class II epitopes based on the screening of engineered reporter cells expressing novel pMHC-TCR (MCR) hybrid molecules carrying cDNA-derived peptides. This technology identifies natural epitopes of CD4+ T cells in an unbiased and efficient manner and allows detailed analysis of TCR cross-reactivity that provides recognition patterns beyond discrete peptides. We determine the cognate peptides of virus- and tumor-specific T cells in mouse disease models and present a proof of concept for human T cells. Furthermore, we use MCR to identify immunogenic tumor neo-antigens and show that vaccination with a peptide naturally recognized by tumor-infiltrating lymphocytes efficiently protects mice from tumor challenge. Thus, the MCR technology holds promise for basic research and clinical applications, allowing the personalized identification of T cell-specific neo-antigens in patients.

Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLβ3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4–expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1–specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

Method Development for the Detection of Human Myostatin by High-Resolution and Targeted Mass Spectrometry.

Myostatin, a highly conserved secretory protein, negatively regulates muscle development, affecting both the proliferation and differentiation of muscle cells. Proteolytic processing of the myostatin precursor protein generates a myostatin pro-peptide and mature protein. Dimerization of the mature myostatin protein creates the active form of myostatin. Myostatin dimer activity can be inhibited by noncovalent binding of two monomeric myostatin pro-peptides. This ability for myostatin to self-regulate as well as the altered expression of myostatin in states of abnormal health (e.g., muscle wasting) support the need for specific detection of myostatin forms. Current protein detection methods (e.g., Western blot) rely greatly on antibodies and are semiquantitative at best. Tandem mass spectometry (as in this study) provides a highly specific method of detection, enabling the characterization of myostatin protein forms through the analysis of discrete peptides fragments. Utilizing the scheduled high-resolution multiple reaction monitoring paradigm (sMRMHR; AB SCIEX 5600 TripleTOF) we identified the lower limit of quantitation (LLOQ) of both mature (DFGLDCDEHSTESR) and pro-peptide regions (ELIDQYDVQR) as 0.19 nmol/L. Furthermore, scheduled multiple reaction monitoring (sMRM; AB SCIEX QTRAP 5500) identified a LLOQ for a peptide of the pro-peptide region (LETAPNISK) as 0.16 nmol/L and a peptide of the mature region (EQIIYGK) as 0.25 nmol/L.

MOLECULAR DYNAMICS SIMULATIONS OF PEPTIDE–SWCNT INTERACTIONS RELATED TO ENZYME CONJUGATES FOR BIOSENSORS AND BIOFUEL CELLS

With the demonstration of direct electron transfer between the redox active prosthetic group, flavin adenine dinucleotide (FAD), of glucose oxidase (GOx) and single-walled carbon nanotubes (SWCNT), there has been growing interest in the fabrication of CNT-enzyme supramolecular constructs that control the placement of SWCNTs within the tunneling distance of co-factors for enhanced electron transfer efficiency in generation-3 biosensors and advanced biofuel cells. These conjugate systems raise a series of questions such as: which peptide sequences within the enzymes have high affinity for the SWCNTs? And, are these high affinity sequences likely to be in the vicinity of the redox-active co-factor to allow for direct electron transfer? Phage display has recently been used to identify specific peptide sequences that have high affinity for SWCNTs. Molecular dynamics simulations were performed to study the interactions of five discrete peptides with (16,0) SWCNT in explicit water as well as with graphene. From the progression of the radius of gyration, Rg, the peptides studied were concertedly adsorbed to both the SWCNT and graphene. Peptide properties calculated using individual amino acid values, such as hydrophobicity indices, did not correlate with the observed adsorption behavior as quantified by Rg, indicating that the adsorption behavior of the peptide was not based on the individual amino acid residues. However, the Rg values, reflective of the physicochemical embrace of the surface (SWCNT or graphene) had a strong positive correlation with the solubility parameter, indicating concerted, cooperative interaction of peptide segments with the materials. The end residues appear to dominate the progression of adsorption regardless of character. Sequences identified by phage display share some homology with key enzymes (GOx, lactate oxidase and laccase) used in biosensors and enzyme-based biofuel cells. These analogous sequences appear to be buried deep within the shell of fully folded proteins and as such are expected to be close to the redox-active prosthetic group.

Biophysical mimicry of lung surfactant protein B by random nylon-3 copolymers.

Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Structure−activity Relationships among Random Nylon-3 Copolymers That Mimic Antibacterial Host-Defense Peptides

Host-defense peptides are natural antibiotics produced by multicellular organisms to ward off bacterial infection. Since the discovery of these molecules in the 1980s, a great deal of effort has been devoted to elucidating their mechanisms of action and to developing analogues with improved properties for possible therapeutic use. The vast majority of this effort has focused on materials composed of a single type of molecule, most commonly a peptide with a specific sequence of alpha-amino acid residues. We have recently shown that sequence-random nylon-3 copolymers can mimic favorable properties of host-defense peptides, and here we document structure-activity relationships in this polymer family. Although the polymers are heterogeneous in terms of subunit order and stereochemistry, these materials display structure-activity relationships comparable to those that have been documented among host-defense peptides and analogous synthetic peptides. Previously such relationships have been interpreted in terms of a specific and regular folding pattern (usually an alpha-helix), but our findings show that these correlations between covalent structure and biological activity do not require the adoption of a specific or regular conformation. In some cases our observations suggest alternative interpretations of results obtained with discrete peptides.

A test for randomness in peptides isolated from a thermal polyamino acid

A thermal polymer of glutamic acid, glycine, and tyrosine was fractionated on Sephadex and on paper. Discrete peptides were found. Of those tripeptides containing tyrosine, only pyroglu-tamyltyrosylglycine and pyroglutamylglycyltyrosine were identified; the sequences were assigned by classical techniques and by mass spectrometry of the permethylated peptides. The amount of these two tripeptides found is 19 times as great as that calculated on the a priori basis of randomness. The presence of two tyrosine-containing tripeptides, in contrast to 30 theoretical isomers, is also highly nonrandom. As in other studies, thermal polymerization of amino acids is constrained, not random.

Inhibition of IFN-α/β signaling by two discrete peptides within measles virus V protein that specifically bind STAT1 and STAT2

The V protein of measles virus (MV-V) is a potent inhibitor of IFN-α/β signaling pathway. We previously reported that when physically dissociated, the N-terminal and C-terminal regions of MV-V (PNT and VCT, respectively) could independently impair signal transduction. The PNT region inhibited IFN-α/β signaling by interacting with at least two components of this pathway: Jak1 and STAT1. Here we report a direct interaction between the VCT of MV-V and STAT2, a third component of IFN-α/β transduction machinery. This interaction with STAT2 is carried by the cysteine-constrained peptide of 49 amino acids localized in the VCT region, and is essential to the inhibition of IFN-α/β signaling. In parallel, we also mapped STAT1 binding site in the PNT region and identified a minimal peptide of only 11 amino acids. IFN-α/β signaling was impaired in human cells treated with this MV-V peptide fused to a cell-penetrating sequence. Finally, we show that signaling downstream of IFN-λ, a recently identified cytokine that also relies on STAT1, STAT2 and Jak1 to transduce, is blocked by MV-V. Altogether, our results illustrate how a single viral protein has evolved to achieve a robust inhibition of the antiviral response by interacting with several signaling molecules.

Gene-specific inhibition of breast carcinoma in BALB-neuT mice by active immunization with rat Neu or human ErbB receptors.

Employing the transgenic BALB-neuT mouse tumor model, we explored the in vivo biologic relevance of immunocompetent epitopes shared among the four ErbB receptors. The outcome of neu-mediated tumorigenesis was compared following vaccination with isogeneic normal rat ErbB2/Neu (LTR-Neu) or xenogeneic human ErbB receptors (LTR-EGFR, LTR-ErbB2, LTR-ErbB3 and LTR-ErbB4), each recombinantly expressed in an NIH3T3 murine cell background. Vaccination using rat LTR-Neu at the stage of atypical hyperplasia potently inhibited neu-mediated mammary tumorigenesis. Moreover, all human ErbB receptors specifically interfered with tumor development in BALB-neuT mice. Relative increase in tumor-free survival and reduction in tumor incidence corresponded to structural similarity shared with the etiologic neu oncogene, as rat orthologue LTR-Neu proved most effective followed by the human homologue LTR-ErbB2 and the other three human ErbB receptors. Vaccination resulted in high titer specific serum antibodies, whose tumor-inhibitory effect correlated with cross-reactivity to purified rat Neu extracellular domain in vitro. Furthermore, a T cell response specific for peptide epitopes of rat Neu was elicited in spleen cells of mice immunized with LTR-Neu and was remotely detectable for discrete peptides upon vaccination with LTR-ErbB2 and LTR-EGFR. The most pronounced tumor inhibition by LTR-Neu vaccination was associated with leukocyte infiltrate and tumor necrosis in vivo, while immune sera specifically induced cytotoxicity and apoptosis of BALB-neuT tumor cells in vitro. Our findings indicated that targeted inhibition of neu oncogene-mediated mammary carcinogenesis is conditional upon the immunization schedule and discrete immunogenic epitopes shared to a variable extent by different ErbB receptors.

Interpretation of Shotgun Proteomic Data

The shotgun proteomic strategy based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching has become the method of choice for identifying proteins in most large scale studies. However, the peptide-centric nature of shotgun proteomics complicates the analysis and biological interpretation of the data especially in the case of higher eukaryote organisms. The same peptide sequence can be present in multiple different proteins or protein isoforms. Such shared peptides therefore can lead to ambiguities in determining the identities of sample proteins. In this article we illustrate the difficulties of interpreting shotgun proteomic data and discuss the need for common nomenclature and transparent informatic approaches. We also discuss related issues such as the state of protein sequence databases and their role in shotgun proteomic analysis, interpretation of relative peptide quantification data in the presence of multiple protein isoforms, the integration of proteomic and transcriptional data, and the development of a computational infrastructure for the integration of multiple diverse datasets.

Substrate Specificity and Novel Selective Inhibitors of TNF-α Converting Enzyme (TACE) from Two-Dimensional Substrate Mapping

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

Statistical and Computational Methods for Comparative Proteomic Profiling Using Liquid Chromatography-Tandem Mass Spectrometry

The combined method of LC-MS/MS is increasingly being used to explore differences in the proteomic composition of complex biological systems. The reliability and utility of such comparative protein expression profiling studies is critically dependent on an accurate and rigorous assessment of quantitative changes in the relative abundance of the myriad of proteins typically present in a biological sample such as blood or tissue. In this review, we provide an overview of key statistical and computational issues relevant to bottom-up shotgun global proteomic analysis, with an emphasis on methods that can be applied to improve the dependability of biological inferences drawn from large proteomic datasets. Focusing on a start-to-finish approach, we address the following topics: 1) low-level data processing steps, such as formation of a data matrix, filtering, and baseline subtraction to minimize noise, 2) mid-level processing steps, such as data normalization, alignment in time, peak detection, peak quantification, peak matching, and error models, to facilitate profile comparisons; and, 3) high-level processing steps such as sample classification and biomarker discovery, and related topics such as significance testing, multiple testing, and choice of feature space. We report on approaches that have recently been developed for these steps, discussing their merits and limitations, and propose areas deserving of further research.

Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting.

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca(2+)-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca(2+) concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca(2+) sensitive. Ca(2+)titration isotherms monitoring the oxidation within residues 49-72 (within subdomain S1), 121-135 (S1), 162-166 (S2), and 722-748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca(2+) concentration of 1-5 microM that is competent for capping and severing activity. A second structural transition with a midpoint of approximately 60-100 microM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical "latch" of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca(2+) transition. However, residues within S4 that are part of an extended beta-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca(2+) concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.

EVIDENCE THAT HAX-1 IS AN INTERLEUKIN-1α N-TERMINAL BINDING PROTEIN

During studies aimed at understanding the function of the N-terminal peptide of interleukin-1α (IL-1 NTP, amino acids 1–112), which is liberated from the remainder of IL-1α during intracellular processing, we identified by yeast two-hybrid analysis a putative interacting protein previously designated as HAX-1. In vitro binding studies and transient transfection experiments confirmed that HAX-1 can associate with the IL-1 NTP. HAX-1 was first identified as a protein that associates with HS1, a target of non-receptor protein tyrosine kinases within haematopoietic cells. Recent data have also revealed interactions between HAX-1 and three disparate proteins, polycystin-2 (derived from the PKD2 gene), a protein linked to polycystic kidney disease, cortactin, and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP). Sequence analysis of different HAX-1 binding domains revealed a putative consensus binding motif that is present in various intracellular proteins. Overlapping peptides comprising the IL-1 NTP were synthesized, and binding experiments revealed that discrete peptides were capable of interacting with HAX-1. HAX-1 may serve to retain the IL-1 NTP in the cytoplasm, and complex formation between the IL-1 NTP and HAX-1 may play a role in motility and/or adhesion of cells.

Molecular and Functional Characterization of Protein 4.1B, a Novel Member of the Protein 4.1 Family with High Level, Focal Expression in Brain

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Selection of phage-display peptides that bind to cucumber mosaic virus coat protein

Several discrete peptides that bind specifically to the coat protein of cucumber mosaic virus (CMV) were isolated from a diverse phage library displaying random nonapeptides on the major coat protein VIII. Enrichment was shown by polyclonal phage enzyme linked immunosorbent assay (ELISA) after three rounds of selection. Sequencing of the genes encoding 10 of these peptides revealed an absence of any conserved motifs, although nine of them contained a high proportion of proline residues. Some of the selected peptides were displayed at the N-terminus of thioredoxin and expressed in the cytoplasm of Escherichia coli. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CMV and CMV present in crude leaf extracts from infected plants. By dot blot analysis, a thioredoxin–peptide fusion could readily detect as little as 5 ng of CMV. The peptides did not bind to other plant viruses. These peptides have been shown to be specific and highly sensitive tools in the detection of CMV and, as well as their diagnostic potential, they could form the basis for a novel disease resistance strategy.