A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16-21 and ubiquitin 52-57 fragments, and second designed to mimic the ubiquitin 5-13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.     

Immunosuppressory activity of ubiquitin fragments containing retro-RGD sequence

A peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) (U50-59) possesses a very high immunosuppressory activity, comparable to that of cyclosporine, both in the cellular and humoral immune responses. We found that the pentapeptide DGRTL (U52-56) is the shortest, effective immunosuppressory fragment of ubiquitin, although its potency is weaker than that of U50-59. Replacement of each consecutive residue with alanine in U52-56 allowed identification of essential amino acids involved in the immunosuppression. We also evaluated the roles of its N- and C-terminal groups by their acetylation and/or amidation, respectively. The active sequence is located in the external loop of the molecule and therefore it may serve as an important functional epitope for intermolecular binding. Based on the crystal structure of ubiquitin molecule, we designed and synthesized the cyclic analogue with a restricted conformation, cyclo(Glt-Gln-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Lys)-NH2 (Glt = glutaryl) by reacting the C-terminal Lys side chain with the glutarylated N-terminus. The peptide was designed to mimic the ubiquitin(48-59) loop, in order to obtain the ligand that may interact with hypothetical receptors of the loop. The cyclization product selectively but strongly suppresses the cellular immune response. The results indicate that the 48-59 loop may serve as an important functional epitope in the ubiquitin molecule for intermolecular binding.     

Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides

Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells . The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides.     

Degradation of fibrin-β amyloid co-aggregate: A novel function attributed to ubiquitin

Human placental extract contains numerous bioactive components that are effective wound healing, antimicrobial and anti-inflammatory agents. During our investigation on the therapeutic potency of human placental extract, we have purified ubiquitin-like molecules that showed strong fibrino(geno)lytic activity. Further investigation confirmed similar potency of ubiquitin purified from adult human erythrocyte. Additionally, ubiquitin efficiently degraded disordered amyloid β 42 peptide (Aβ42) aggregate and fibrin-Aβ42 co-aggregate in vitro and reduced co-aggregate induced cytotoxicity in SH-SY5Y human neuroblastoma cells as compared to plasmin. Ubiquitin also degraded abnormal co-aggregates of fibrin with other plasma proteins such as fibronectin, albumin, lysozyme, tranthyretin and α-synuclein. To elucidate the mechanism of degradation, synthetic peptides (ADG, GKT, DQQ, QRL, LIF, AGK, HLVL) derived from ubiquitin template as well as synthetic ubiquitin (8565.32?Da) were employed. Synthetic ubiquitin completely degraded preformed Aβ 42 aggregate and fibrin-Aβ42 co-aggregate, whereas, the smaller synthetic peptides showed varying degrees of degradation. These observations attribute a novel function of ubiquitin that may be used for degrading abnormal fibrin clots in human body. Thorough investigation might unfold a novel molecular mechanism of ubiquitin in protein homeostasis.     

HLA‐DQ7 β1 and β2 derived peptides as immunomodulators

Modulation of protein–protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic β‐regions, exposed to the microenvironment, of the modeled HLA‐DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132–141 and 58–67, located at the β₁ and β₂ domains of HLA‐DQ7, respectively, were tested for their involvement in the interactions with CD4⁺ T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA‐DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA‐DQ7 58–67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58–67 segment is located at the β₁ region of HLA‐DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58–67 peptide could accommodate the association between T‐cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co‐stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein‐labeled 58–67 peptide and by the fact that it is an immunostimulator at low concentration. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Ubiquitin trafficking to the lysosome: keeping the house tidy and getting rid of unwanted guests

Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens.(1-5) In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.     

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.     

Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N‐terminus to N‐terminus, C‐terminus to C‐terminus, and N‐terminus to C‐terminus (head‐to‐tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α‐amino groups of the C‐terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head‐to‐tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of degradation on binding properties and biological activity of endomorphin 1

The recently-isolated endogenous peptide endomorphin 1 has high affinity for the mu opioid receptor and plays an important role in analgesia. Several of its degradation products have been isolated from the central nervous system. Degradation products present structural similarities and may influence the receptor binding properties and biological activity of the parent compound. Therefore, we investigated how degradation of endomorphin 1 might influence ligand binding to the mu opioid receptor, the consequent activation of G proteins and its antinociceptive effect. Both N- and C-terminal truncation of endomorphin 1 resulted in peptides presenting considerably lower opioid receptor binding potency. None of these peptides had an effect on GTP binding, nor was able to produce analgesia, suggesting that degradation destroys the biological activity of endomorphin 1.     

Antimicrobial activity of human islet amyloid polypeptides: an insight into amyloid peptides' connection with antimicrobial peptides

Abstract Human islet amyloid polypeptide (hIAPP) shows an antimicrobial activity towards two types of clinically relevant bacteria. The potency of hIAPP varies with its aggregation states. Circular dichroism was employed to determine the interaction between hIAPP and bacteria lipid membrane mimic. The antimicrobial activity of each aggregate species is associated with their ability to induce membrane disruption. Our findings provide new evidence revealing the antimicrobial activity of amyloid peptide, which suggest a possible connection between amyloid peptides and antimicrobial peptides.     

Ubiquitin Trafficking to the Lysosome: Keeping the House Tidy and Getting Rid of Unwanted Guests

Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens. In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.

Addendum to:

Lysosomal Killing of Mycobacterium Mediated by Ubiquitin-Derived Peptides is Enhanced by Autophagy

S. Alonso, K. Pethe, D.G. Russell and G.E. Purdy

Proc Natl Acad Sci USA 2007; 104:6031-6     

Synthesis of IB-01212 by multiple N-methylations of peptide bonds

There are many natural peptides with multiple N-methylamino acids that exhibit potent attractive biological activities. N-methylation of a peptide bond(s) is also one of the standard approaches in medicinal chemistry of bioactive peptides, to improve the potency and physicochemical properties, especially membrane permeability. In this study, we investigated a facile synthesis process of N-methylated peptides via simultaneous N-methylation of several peptide bonds in the presence of peptide bonds that were not to be methylated. As a model study, we investigated the synthesis of the antiproliferative depsipeptide, IB-01212. We used a pseudoproline to protect the non-methylated peptide bond during a simultaneous N-methylation with MeI–Ag₂O. Using further manipulations including a dimerization/cyclization process, IB-01212 and its derivatives were successfully synthesized. A preliminary structure–activity relationship study demonstrated that the symmetric structure contributed to the potent cytotoxic activity of IB-01212.     

Small molecules enhance the potency of natural antimicrobial peptides

The skin-associated microbiome plays an important role in general well-being and in a variety of treatable skin conditions. In this regard, endogenous antimicrobial peptides have both a direct and indirect role in determining the composition of the microbiota. We demonstrate here that certain small molecular species can amplify the antimicrobial potency of naturally occurring antimicrobial peptides. In this study, we have used niacinamide, a form of vitamin B3 naturally found in foods and widely used in cosmetic skincare products, and two of its structural analogs, to investigate their cooperativity with the human antimicrobial peptide LL37 on the bacterium Staphylococcus aureus. We observed a clear synergistic effect of niacinamide and, to some extent, N-methylnicotinamide, whereas isonicotinamide showed no significant cooperativity with LL37. Adaptively biased molecular dynamics simulations using simplified model membrane substrates and single peptides revealed that these molecules partition into the headgroup region of an anionic bilayer used to mimic the bacterial membrane. The simulated effects on the physical properties of the simulated model membrane are well correlated with experimental activity observed in real biological assays despite the simplicity of the model. In contrast, these molecules have little effect on zwitterionic bilayers that mimic a mammalian membrane. We conclude that niacinamide and N-methylnicotinamide can therefore potentiate the activity of host peptides by modulating the physical properties of the bacterial membrane, and to a lesser extent through direct interactions with the peptide. The level of cooperativity is strongly dependent on the detailed chemistry of the additive, suggesting an opportunity to fine-tune the behavior of host peptides.     

Recognized sequence and conformation in design of linear peptides as a competitive inhibitor for HMG-CoA reductase

This study is an attempt to develop a simple search method for lead peptide candidates, which include constrained structures in a recognized sequence, using the design of a competitive inhibitor for HMG-CoA reductase (HMGR). A structure-functional analysis of previously synthesized peptides proposes that a competitive inhibitory peptide can be designed by maintaining bioactive conformation in a recognized sequence. A conformational aspect of the structure-based approach was applied to the peptide design. By analysis of the projections obtained through a principle component analysis (PCA) for short linear and cyclic peptides, a head-to-tail peptide cycle is considered as a model for its linear analogy. It is proposed that activities of the linear peptides based on an identical amino acid sequence, which are obtained from a less flexible peptide cycle, would be relatively higher than those obtained from more flexible cyclic peptides. The design criterion was formulated in terms of a 'V' parameter, reflecting a relative deviation of an individual peptide cycle from an average statistical peptide cycle based on all optimized structures of the cyclic peptides in set. Twelve peptide cycles were selected for the peptide library. Comparing the calculated 'V' parameters, two cyclic peptides (GLPTGG and GFPTGG) were selected as lead cycles from the library. Based on these sequences, six linear peptides obtained by breaking the cycle at different positions were selected as lead peptide candidates. The linear GFPTGG peptide, showing the highest inhibitory activity against HMGR, increases the inhibitory potency nearly tenfold. Kinetic analysis reveals that the GFPTGG peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 6.4 +/- 0.3 microM. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

Modulation of antimicrobial potency of human cathelicidin peptides against the ESKAPE pathogens and in vivo efficacy in a murine catheter-associated biofilm model

Antimicrobial peptides are essential components of innate immune systems that protect hosts from infection. They are also useful candidates for developing a new generation of antibiotics to fight antibiotic-resistant pathogens. Human innate immune peptide LL-37 can inhibit biofilm formation, but suffers from high cost due to a long peptide length and rapid protease degradation. To improve the peptide, we previously identified the major active region and changed the peptide backbone structure. This study designed two families of new peptides by altering peptide side chains. Interestingly, these peptides displayed differential potency against various ESKAPE pathogens in vitro and substantially reduced hemolysis. Further potency test in vivo revealed that 17tF-W eliminated the burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 in both mouse-embedded catheters and their surrounding tissues. In addition, peptide treatment suppressed the level of chemokine TNFα, and boosted the levels of chemokines MCP-1, IL-17A and IL-10 in the surrounding tissues of the infected catheter embedded in mice. In conclusion, we have designed a set of new LL-37 peptides with varying antimicrobial activities, opening the door to potential topical treatment of infections involving different drug-resistant pathogens.     

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.     

Interactions of caveolin-1 scaffolding and intramembrane regions containing a CRAC motif with cholesterol in lipid bilayers

Caveolin-1 is a major structural protein of caveolae and specifically binds cholesterol (Chol). The caveolin scaffolding domain is thought to be involved in caveolin–Chol interaction through the sequence V94-T-K-Y-W-F-Y-R101, a motif that matches a cholesterol recognition amino-acid consensus (CRAC). In the present work, three CRAC-containing peptides, corresponding to caveolin-1 94–101, 82–101 and 93–126, were tested to study the role of the CRAC motif in the caveolin–Chol interaction in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD). The Y97I substituents of the three peptides and one peptide segment corresponding to caveolin-1 101–126 that excludes the CRAC motif were also tested for comparison. Our results showed the potency of these CRAC-containing peptides in sequestering Chol into domains and the enhanced role of the intramembrane domain and scaffolding domain for the potency. Of the three CRAC-containing peptides, the peptide 93–126 was particularly effective in promoting Chol segregation, while the peptide 82–101 was less potent in promoting the formation of domains than the peptide 93–126, but was more potent than the peptide 94–101. The domain partition of DPPC/Chol bilayers was not observed in the presence of the peptide 101–126, in contrast to the case in the presence of the peptide 93–126 at the same concentrations of peptide and Chol. The potency of the CRAC motif in Chol segregation was lowered by the Y97I mutation. The difference in structure may be a factor that contributes to different effects of these peptides on the distribution of Chol in the lipid membrane.     

A tridecapeptide possesses both antimicrobial and protease-inhibitory activities

A 13-residue synthetic peptide (Rev4) was designed based on indolicidin, an antimicrobial peptide from bovine neutrophils. The synthetic peptide retains high antimicrobial activity. When tested for its stability in tobacco leaf extracts, Rev4 was highly stable compared to another antimicrobial peptide, magainin. When mixed with Rev4, magainin was protected from degradation by the leaf extract. Our results show that Rev4 is a potent protease inhibitor which selectively inhibits three out of four different types of proteases. Four other synthetic peptides were tested and the results were suggestive of no correlation between their antimicrobial and protease inhibitory activities.     

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full‐length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein‐2 (BMP‐2), transforming growth factor‐beta 1 (TGF‐β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full‐factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1550–1557, 2013