archives@tulane.edu / During the 2013-2016 West African outbreak, severe gastrointestinal symptoms were common in Ebola patients and associated with poor outcome. The efficient spread of Ebola virus (EBOV) via vomitus and diarrheal fluids, which contain high concentrations of virus, likely contributed to the scale of the outbreak. The delta-peptide is a conserved product of post-translational processing of the abundant EBOV soluble glycoprotein (sGP). Here, the murine ligated ileal loop model, which is well-established for the study of diarrheal disease, was used to demonstrate that delta-peptide is a potent enterotoxin. Dramatic intestinal fluid accumulation peaked at 9-12 hours following injection of biologically relevant amounts of delta-peptide into ileal loops, along with gross destruction of the villous architecture, loss of goblet cell polysaccharides, and secretion of pro-inflammatory cytokines. Transcriptomic analyses showed that delta-peptide triggers immune and cell survival responses. Delta-peptide may contribute greatly to EBOV-induced gastrointestinal pathology in humans. These findings demonstrate that the EBOV delta-peptide is an enterotoxic viroporin and may be categorized as a novel virulence factor. We then hypothesized that the delta-peptide may also be a druggable target to explore a new avenue for novel EBOV therapeutics, which are direly needed. An unconventional coupling strategy was employed to conjugate a modified version of the 23-residue delta-peptide to a carrier protein Keyhole Limpet Hemocyanin (KLH) in order to immunize rabbits so that they may generate high-affinity binding antibodies against the delta-peptide. Antisera was collected from the rabbits after regular immunizations and the IgG fraction of the antisera demonstrated binding and recognition against several delta-peptide variants confirmed by Western blotting and ELISAs. We then used this knowledge to determine therapeutic index in vitro by testing the antibody against the delta-peptide in the context of synthetic PC-PG vesicles and CHO cells. The purified IgG was then tested against the peptide in vivo to determine therapeutic efficacy by returning to the mouse model of diarrheal pathology mentioned previously. In a small pilot experiment, we were able to successfully block the enterotoxic activity of the delta-peptide and did not observe gastrointestinal distress in the mice that were treated with the peptide and antibody together versus just the peptide alone, signifying that the delta-peptide is a druggable target and may reveal a new therapeutic avenue against EBOV pathogenesis. / 1 / Shantanu Guha

Hydrogels have been used for many applications, including as a mimic for the extracellular matrix (ECM) in cell culture. For example, a hydrogel containing protease-sensitive substrates can be used to create an environment that cells can modify via enzymatic degradation. In this study, we propose combining traditional hydrogels for cell culture with “cryptic” site that bury proteolytically cleavable peptide sequences using complex coacervation. Here, the goal is to take advantage of the phase separation of coacervates to protect the cleavable peptide against degradation until acted upon by a mechanical force, such as those generated by adherent cells. To this end, we studied the encapsulation of chymotrypsin as a model protease into our coacervate system and investigated the effect of incorporation into the coacervate on its activity. We have also synthesized a peptide containing cleavable site for both chymotrypsin as our model protease and more biologically relevant matrix metalloproteinases (MMPs). Future efforts will look to incorporate this peptide into both coacervate and hydrogel and test the level of cryptic response.

Various alkaline proteases derived from skeletal muscle have been described by a number of researchers and have been purified to varying degrees. Such alkaline proteases may play an important role in the metabolism of myofibrillar and other muscle proteins and as such deserve to be fully characterised. In this study, a major myofibrillar alkaline protease was purified from rat skeletal muscle. The enzyme degraded both denatured casein and azocasein and had a pH optimum of 9,0. The molecular mass was 32 250 ± 650. The presence of a second, minor alkaline protease was demonstrated using three different separation techniques as well as by inhibitor studies. The major protease was insensitive to inhibition by pepstatin and leupeptin, whilst 90 % of the activity was expressed in the presence of 2 mM EGTA. A moderate degree of inhibition was observed in the presence of soybean trypsin inhibitor and the protease was markedly sensitive to chymostatin. A similar alkaline protease was partially purified from rat cardiac muscle using the same purification procedure. Incubation of washed myofibrils in the presence of sodium pyrophosphate released a factor into the supernatant, the removal of which facilitated the separation of myofibrillar alkaline protease from the myofibrils. The factor appeared to be necessary for binding of the alkaline protease to the myofibrillar proteins but its removal did not disrupt the binding of proteolytic activity already attached to the myofibrillar proteins. An inhibitor of myofibrillar alkaline protease was demonstrated which is, in principle, capable of playing an important regulatory role in controlling the activity of these enzymes and thereby of myofibrillar protein catabolism.

Cancer is one of the leading causes of death in the United States. Monoclonal antibody drugs became one of the commercially and clinically successful drugs for many diseases. Among the monoclonal antibodies, T cell-dependent bispecific antibodies directly target tumor cells for cancer treatment, they kill tumor cells by activating T cells through binding to the CD3 (cluster of differentiation 3) receptor on T cells. Many anti-CD3 antibodies bind to the surface exposed CD3 γ, δ, ε subunits for T cell activation. SP34, an anti-CD3ε antibody, specifically binds to the first 27 amino acids of CD3ε. Synthesis of CD3ε peptides proofed to be difficult due to its hydrophobic nature and presence of an N-terminal glutamine that caused many side reactions resulting in very poor peptide quality and purity. For some commercial full-length CD3ε proteins it is unclear whether N-terminal glutamine is present or absent. In cases where N-terminal glutamine is present it is modified to pyroglutamic acid. To study the SP34-CD3ε interaction a reliable and defined source of CD3ε peptide and peptide variants is required. By utilizing the SUMO (Small Ubiquitin-related Modifier) system from yeast, CD3ε1-27 amino acid and a truncated version 2-27 amino acid peptide are expressed in E. coli cells with an SMT3 (Mitotic Fidelity Gene 3) tag. Subsequently, SMT3 tag is removed with SUMO protease and the resulting peptide is further purified. This novel in vitro approach results in high yields of non-modified peptides with great purity (>95%).

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Cell adhesion and growth are central issues in the otherwise promising method of endothelialization of materials for improving blood compatibility. To improve cell adhesion to biomaterial surfaces, surface modification with cell adhesion peptides is often used. In particular, arginine-glycine-aspartic acid (RGD), long recognized as a peptide sequence that plays and important role in cell adhesion, has been covalently attached to surfaces to enhance cell adhesion. In this work, the effect of the orientation of the cell adhesion peptide immobilized on the surface was studied through using gold surfaces, which can be readily modified with thiols and sulfur-containing groups. Peptide orientation was controlled by the placement of the cysteine (C) at either the C- or N-terminus, Two cell adhesion peptides, the non-specific RGD and more endothelial cell specific REDV (arginine-glutamic acid-aspartic acid-valine), were studied for their effect on the surface chemical and biological properties, including effects on the interactions with the endothelial cell line ECV304. Vitronectin adsorption to the modified surfaces was specifically examined as a possible reason for differences noted. The results suggest that peptide orientation plays an important role in the interactions of cells and proteins to the modified surfaces. Peptides with the cysteine at the N-terminus showed increased adhesion of endothelial cells from the ECV304 line, with the greatest adhesion noted consistently on the CREDV-modified surfaces. Differences in surface chemistry as evaluated by x-ray photoelectron spectroscopy were also found higher levels of bonded peptide when the thiol-containing cysteine was in the N-terminal position. These results suggest that the secondary structure of the peptide can be used to enhance or to limit its reaction with the surface. Furthermore, while cell adhesion was noted during culture in the absence of serum, significant increases in the numbers of adherent cells were noted on all surfaces when the cells were grown in the presence of serum. Immunoblotting and culture with antibodies demonstrated that this increase in the adhesion of the cells is likely mediated primarily by the cell adhesion peptide vitronectin. / Thesis / Master of Engineering (ME)

Gold, as a chemically inert metal, does not form a stable oxide, but has strong specific interactions with sulfur functions. It has been found that thiols or disulfides can chemisorb to gold under mild conditions (room temperature), and form densely packed monolayers on the gold surface due to the high density of binding sites (gold atoms). Thus, it is possible to form closely packed and stable monolayers of thiolates containing desirable bioactive moieties. Thiol-gold chemistry may therefore be considered as a potentially important tool in the surface modification of materials for biomedical applications. In order to develop surfaces with antithrombogenic properties, a number of thrombin inhibitors (heparin, hirudin and PPACK) have been bonded or immobilized to the material surfaces. In the present study, a series of short peptides, cys-pro-arg (CPR), cys-phe-pro-arg (CFPR) and cys-(D)-phe-pro-arg (C[D]FPR), analogues of PR and FPR respectively, were chosen as potential thrombin inhibitors to attach to gold-coated polyurethane surfaces via the cysteine residue. Their inhibitory activity against thrombin was verified by a chromogenic substrate assay. C(D)FPR, showed a relatively high level of inhibition activity. The surfaces were characterized by water contact angle, XPS, AFM, SEM, ellipsometry, and infrared reflection-absorption spectroscopy, and the adsorption of thrombin from buffer and modified plasma was investigated. It was found that the peptide modified gold surfaces adsorbed significantly more thrombin than the unmodified control surfaces. The C(D)FPR-modified gold surface showed the highest thrombin adsorption both from buffer and plasma. This results is in accord with previous studies showing that the D form of phenylalananine in the FPR peptide creates a favourable site geometry for binding to thrombin. The activity of thrombin adsorbed on these peptide modified gold surfaces was also investigated using a chromogenic substrate assay. Inhibition of adsorbed thrombin was demonstrated, and the C(D)FPR surface showed the strongest inhibitory activity. The presence of thrombin on the peptide surfaces following exposure to modified plasma was verified by elution of proteins and identification of thrombin in the eluate. Probing of the eluates with antibodies to 25 plasma proteins showed that the peptide surfaces are relatively non-adsorptive, suggesting they have some degree of selectivity for thrombin binding. / Thesis / Master of Engineering (ME)

Absorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported. / Master of Science