Essam Refai

Glucagon‐like peptide 1 (GLP‐1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half‐life (t_1/2 < 2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half‐life extension and sustained release have been explored. In the present study, conjugates between a GLP‐1 analogue and a 5 kDa albumin‐binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP‐1‐ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose‐dependent, showing no significant difference between the three different GLP‐1‐ABD conjugates and unconjugated GLP‐1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP‐1‐ABD conjugates promising GLP‐1 receptor agonists expected to show extended in vivo half‐life. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 252–259, 2014.

Transthyretin (TTR) is a functional protein in the pancreatic β-cell. It promotes insulin release and protects against β-cell death. We now demonstrate by ligand blotting, adsorption to specific magnetic beads, and surface plasmon resonance that TTR binds to glucose-regulated proteins (Grps)78, 94, and 170, which are members of the endoplasmic reticulum chaperone family, but Grps78 and 94 have also been found at the plasma membrane. The effect of TTR on changes in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) was abolished if the cells were treated with either dynasore, a specific inhibitor of dynamin GTPase that blocks clathrin-mediated endocytosis, or an antibody against Grp78, that prevents TTR from binding to Grp78. The conclusion is that TTR binds to Grp78 at the plasma membrane, is internalized into the β-cell via a clathrin-dependent pathway, and that this internalization is necessary for the effects of TTR on β-cell function.

Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when β cells are exposed to these diabetic sera, apoptosis occurs, an effect abolished by an antibody against apoCIII. We have investigated the BB rat, an animal model that develops a human-like type 1 diabetes, and found that apoCIII was also increased in sera from prediabetic rats. This increase in apoCIII promoted β-cell death. The endogenous levels of apoCIII were reduced by treating prediabetic animals with an antisense against this apolipoprotein, resulting in a significantly delayed onset of diabetes. ApoCIII thus serves as a diabetogenic factor, and intervention with this apolipoprotein in the prediabetic state can arrest disease progression. These findings suggest apoCIII as a target for the treatment of type 1 diabetes.

Antimicrobial peptides are small molecular weight proteins with a large antibacterial spectrum. They can reach high local concentrations in tissues with active inflammation, being largely produced by immunocompetent cells. However, their effect on eukaryotic cells is still unclear. We have, therefore, studied three structurally different antimicrobial peptides (cecropin P1, PR‐39 and NK‐lysin) for their cytotoxic effects on blood mononuclear cells. None of the antimicrobial peptides tested exhibited significant cytotoxic effect on resting lymphocytes isolated either from peripheral blood or from the spleen with the exception of high concentrations (ten times higher than IC100 for Escherichia coli) of NK‐lysin. Activated lymphocytes were, however, more sensitive to the cytotoxic effect of the antimicrobial peptides. Both activated T‐cells and B‐cells were dose dependent sensitive to NK‐lysin while only activated B‐cells but not activated T‐cells were sensitive to PR‐39. Cecropin did not exhibit any cytotoxic effect on activated lymphocytes either. By using several cell lines (3B6, K562, U932 and EL‐4) we were able to show that NK‐lysin has a broad necrotic effect while PR‐39 has a cell specific apoptotic effect dependent on the specifically cellular uptake. In conclusion we show here that antimicrobial peptides are not cytotoxic for the resting eukaryotic cells but can be cytotoxic on activated immune cells through distinct mechanisms of cell death. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

Transthyretin (TTR) is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol, mainly existing as a tetramer in vivo . We now demonstrate that TTR tetramer has a positive role in pancreatic β-cell stimulus-secretion coupling. TTR promoted glucose-induced increases in cytoplasmic free Ca ²⁺ concentration ([Ca ²⁺ ] _i ) and insulin release. This resulted from a direct effect on glucose-induced electrical activity and voltage-gated Ca ²⁺ channels. TTR also protected against β-cell apoptosis. The concentration of TTR tetramer was decreased, whereas that of a monomeric form was increased in sera from patients with type 1 diabetes. The monomer was without effect on glucose-induced insulin release and apoptosis. Thus, TTR tetramer constitutes a component in normal β-cell function. Conversion of TTR tetramer to monomer may be involved in the development of β-cell failure/destruction in type 1 diabetes.

Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variableEscherichia coliactivities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity againstE. colito a larger extent than that againstMycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria.

Antimicrobial peptides are essential effector molecules of the innate immune system. Here we describe the structure, function and diversity of cryptdin-related sequence (CRS) peptides, a large family of antimicrobial molecules. We identified the peptides as covalent dimers in mouse intestinal tissue in amounts comparable to those of Paneth cell–derived enteric α-defensins. CRS peptides caused rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides formed homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract.

We have investigated the in vitro activity of antimicrobial peptides against Mycobacterium tuberculosis using a radiometric method and cfu determinations. PR-39, a proline-arginine-rich antibacterial peptide from porcine leucocytes, was found to be active against drug-susceptible as well as multi-drug-resistant (MDR) clinical isolates of M. tuberculosis. The activity of PR-39 was concentration dependent, with 80% growth inhibition of M. tuberculosis H37Rv at 50 mg/L. The MDR M. tuberculosis strains E1380/94 and P34/95 were less susceptible to PR-39, with 39 and 49% growth inhibition at 50 mg/L peptide, respectively, suggesting a lower susceptibility than strain H37Rv and drug-susceptible clinical isolates. Reduction of counts of M. tuberculosis H37Rv and the MDR M. tuberculosis strain E1380/94 by PR-39 indicated that the growth inhibition seen in the radiometric assay is due to a mycobactericidal effect of the peptide. These observations suggest that antimicrobial peptides may play an important role in host defence against MDR M. tuberculosis.

Neuroblastoma, a neural crest-derived childhood tumor of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. However, the majority of neuroblastomas are diagnosed as metastatic tumors with a poor prognosis despite intensive multimodal therapy. The neuropeptide somatostatin (SOM) has been shown to inhibit neuroblastoma growth and induce apoptosis in vitro. Therapeutic effects of SOM analogues are dependent on tumor expression of high-affinity receptors. In the present study, human neuroblastoma SH-SY5Y cells were grown as xenografts in nude rats. In vivo SOM receptor expression in the xenografts was identified using scintigraphy with 111In-pentetreotide. Rats were randomized to treatment with the long-acting SOM analogue octreotide (10 µg s.c. every 12 h), 13-cis-retinoic acid (4 mg orally every 24 h), or vasoactive intestinal peptide (40 µg s.c. every 24 h) and compared with controls. Tumor volume was assessed every second day and tumor weight after 10-12 d. Octreotide treatment inhibited neuroblastoma growth significantly with reduced tumor volumes at 10 and 12 d compared with untreated controls (mean 3.56 and 4.24 versus 6.48 and 8.01 mL, respectively; p < 0.01). Also, tumor weights after 10-12 d were reduced in octreotide-treated animals (n = 8, median weight 2.90 g, range 1.67-5.57 g) compared with untreated rats (n = 14, 7.54 g, 1.65-10.82 g, p = 0.005). Serum IGF-I decreased significantly over time both in rats treated with octreotide and in untreated controls. It is concluded that treatment with the SOM analogue octreotide may significantly decrease neuroblastoma tumor growth in vivo. Further studies are warranted to establish the role of SOM analogues in the treatment of children with unfavorable neuroblastoma.