Identification of Chiral-Specific Carbon Nanotube Binding Peptides Using a Modified Biopanning Method

Peptides can recognize and selectively bind to a wide variety of materials dependent on both their surface properties and the environment. Biopanning with phage or cell peptide display libraries can identify material-specific binding peptides. However, the limitations with sequence diversity of traditional bacteriophage (phage) display libraries and loss of unique phage clones during the amplification cycles results in a smaller pool of peptide sequences identified. False positive sequences tend to emerge during the biopanning process due to highly proliferating, yet nonspecific, phages. In order to overcome this limitation of traditional biopanning methodology, a modified method using high-throughput next generation sequencing (HTS) was tested to select for unique peptides specific to two types of single wall carbon nanotube (SWNTs) sources with varying diameter distribution and chirality. Here, the process, analysis, and characterization of peptide sequences identified using the modified method is further described and compared to a peptide identified in literature using the traditional method. Selected sequences from this study were incorporated in a SWNT dispersion experiment to probe their selectivity to the nanotube diameter. We show that NHTS can uncover unique binding sequences that might have otherwise been lost during the traditional biopanning method.

Peptide-Based Inhibitors of ADAM and ADAMTS Metalloproteinases

ADAM and ADAMTS are two large metalloproteinase families involved in numerous physiological processes, such as shedding of cell-surface protein ectodomains and extra-cellular matrix remodelling. Aberrant expression or dysregulation of ADAMs and ADAMTSs activity has been linked to several pathologies including cancer, inflammatory, neurodegenerative and cardiovascular diseases. Inhibition of ADAM and ADAMTS metalloproteinases have been attempted using various small molecules and protein-based therapeutics, each with their advantages and disadvantages. While most of these molecular formats have already been described in detail elsewhere, this mini review focuses solely on peptide-based inhibitors, an emerging class of therapeutic molecules recently applied against some ADAM and ADAMTS members. We describe both linear and cyclic peptide-based inhibitors which have been developed using different approaches ranging from traditional medicinal chemistry and rational design strategies to novel combinatorial peptide-display technologies.

tANCHOR: a novel mammalian cell surface peptide display system

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.

DNA Hybridization-Based Differential Peptide Display Identified Potential Osteogenic Peptides

A DNA hybridization-based differential peptide display (DPD) was developed for the screening of phage peptide library to find osteogenic peptides intended to bind to epigenetically induced osteogenic receptors on NIH/3T3 (3T3) cell surface. In the presence of DNA methylation inhibitor of 5-azacytidine (5AZC), an osteoblastic receptor of bone morphogenetic protein (BMP) receptor 1A (BMPR1A) was induced on the cell surface of NIH/3T3 fibroblasts. Cyclic heptamer-displaying phage library was screened against vehicle and 5AZC treated (Tx) 3T3 cells. Antisense oligo against library against library peptide coding DNA of control 3T3 cell bound phages were synthesized to subtract common binders from that of 5AZC-Tx 3T3 cell-bound phages that included 5AZC-induced receptor binders. The library peptide coding regions of conformational receptor binder-subtracted DPD were PCR-amplified and cloned into a plasmid vector specifically designed for short peptide expression. No unique binder was identified when 96 clones were randomly picked from the third round of panning against 5AZC-treated 3T3 cells, suggesting miscellaneous bindings to cell surface proteins. Unique binders showing homology to known function proteins were successfully identified when constitutive receptor binders were subtracted from 5AZC-induced protein binders. Some of identified peptides significantly increased alkaline phosphatase activity in 5AZC-Tx 3T3 cells. DPD can be a useful tool to screen functional peptide bindings to cell surface receptors. Graphic Abstract

Human γδ T cells recognize CD1b by two distinct mechanisms

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.

Predicted Cellular Immunity Population Coverage Gaps for SARS-CoV-2 Subunit Vaccines and their Augmentation by Compact Peptide Sets

Subunit vaccines induce immunity to a pathogen by presenting a component of the pathogen and thus inherently limit the representation of pathogen peptides for cellular immunity based memory. We find that SARS-CoV-2 subunit peptides may not be robustly displayed by the Major Histocompatibility Complex (MHC) molecules in certain individuals. We introduce an augmentation strategy for subunit vaccines that adds a small number of SARS-CoV-2 peptides to a vaccine to improve the population coverage of pathogen peptide display. Our population coverage estimates integrate clinical data on peptide immunogenicity in convalescent COVID-19 patients and machine learning predictions. We evaluate the population coverage of 9 different subunits of SARS-CoV-2, including 5 functional domains and 4 full proteins, and augment each of them to fill a predicted coverage gap.

Kinetically distinct processing pathways diversify the CD8+T cell response to a single viral epitope

The source proteins from which CD8+T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+T cell responses. This dual system of peptide generation enhances CD8+T cell participation in diversifying both antigenicity and the kinetics of peptide display.