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Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins)
Abstract
Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory “domino effect” which may alter cell activation and gene expression.
Peptides, short stretches of amino acids or small proteins that occupy a strategic position between proteins and amino acids, are readily accessible by chemical and biological methods. With ideal properties for forming high-affinity and specific interactions with host target proteins, they have established an important niche in the drug development spectrum complementing small molecule and biological therapeutics. Among the most successful biomedicines in use today, peptide-based drugs show great promise. This, coupled with recent advances in synthetic and nanochemical biology, has led to the creation of tailor-made peptide therapeutics for improved biocompatibility. This review presents an overview of the latest research on pathogen-derived, host-cell-interacting peptides. It also highlights strategies for using peptide-based therapeutics that address cellular transport challenges through the introduction of nanoparticles that serve as platforms to facilitate the delivery of peptide biologics and therapeutics for treating various inflammatory diseases. Finally, this paper describes future perspectives, specific pathogen-based peptides that can enhance specificity, efficiency, and capacity in functional peptide-based therapeutics, which are in the spotlight as new treatment alternatives for various diseases, and also presents verified sequences and targets that can increase chemical and pharmacological value.
Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-deficient (IFNγR−/−) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide S-7 (G305TTASSDTAITLIPR319) induced immune protection, reduced disease severity and improved survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection. Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and hemorrhage, increased mortality and specific modification of gut microbiota. Serp-1 and S-7 reduced pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment efficacy was lost after antibiotic treatments with associated specific changes in the gut bacterial microbiota. In summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection influences gammaherpesviral infection severity and reduces immune modulating therapeutic efficacy.
Serpins are evolutionarily conserved serine protease inhibitor that widely distributed in animals, plants and microbes. In this study, we reported the cloning and functional characterizations of two novel serpin genes, HlSerpin‐a and HlSerpin‐b, from the hard tick Haemaphysalis longicornis of China. Recombinant HlSerpin‐a and HlSerpin‐b displayed protease inhibitory activities against multiple mammalian proteases. Similar to other tick serpins, HlSerpin‐a and HlSerpin‐b suppressed the expression of inflammatory cytokines such as TNF‐α, IL6 and IL‐1β from LPS stimulated mouse bone marrow‐derived macrophages (BMDMs) or mouse bone marrow‐derived dendritic cells (BMDCs). The minimum active region (Reaction Centre Loop) of HlSerpin‐a, named SA‐RCL, showed similar biological activities as HlSerpin‐a in the protease inhibition and immune suppression assays. The immunosuppressive activities of full length HlSerpin‐a and SA‐RCL are impaired in Cathepsin G or Cathepsin B knockout mouse macrophages, suggesting that the immunomodulation function of SA and SA‐RCL are dependent on their protease inhibitory activity. Finally, we showed that both full length HlSerpins and SA‐RCL can relieve the joint swelling and inflammatory response in collagen‐induced mouse arthritis models. These results suggested that HlSerpin‐a and HlSerpin‐b are two functional arthropod serpins, and the minimal reactive peptide SA‐RCL is a potential candidate for drug development against inflammatory diseases.
We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.
Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.
The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N-terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å) and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N-terminus into a pocket equivalent to a hormone binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin’s active site. Insertion of the N-terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin’s active site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT’s hormone binding pocket.
While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a 'first-in-class' therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next generation RCL peptides with improved therapeutic potential that is not reduced when combined with antibiotic treatments. The crystal structure of the cleaved Serp-1 was determined to 2.5 Å resolution, and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII) and alpha-1 antitrypsin (A1AT) and target proteases. Using in silico modeling of Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins due to a higher number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest peptide interaction with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step towards the development of a promising new class of anti-inflammatory serpin-based therapeutic.
The homeostatic chemokine CCL21 plays a pivotal role in lymphocyte homing and compartment localization within the lymph node, and also affects adhesion between immune cells. CCL21's effects are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to antigen presenting cells. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator, and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.Immunology and Cell Biology accepted article preview online, 15 June 2016. doi:10.1038/icb.2016.56.
Serpins regulate coagulation and inflammation, binding serine proteases in suicide inhibitory complexes. Target proteases cleave the serpin reactive center loop (RCL) scissile P1-P1' bond resulting in serpin-protease suicide inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases while mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon gamma receptor deficient mice (IFNγR-/-). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the RCL produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing R or RN, not RM, with 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking R or RN with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b+ monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in PAI-1 serpin deficient transplants. Plaque reduction correlated with mononuclear cell activation..In a separate study Serp-1 peptide, S-7 improved survival in the MHV68 vasculitis model whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.
Lethal viral infections produce widespread inflammation with vascular leak, clotting and bleeding (disseminated intravascular coagulation, termed DIC), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA, uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFNγR) knock-out mice, a model for lethal inflammatory vascultis. Treatment of MHV68 infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68 infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophage. Serp-1 altered gene expression for coagulation and inflammatory responses whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire Ebolavirus in wild type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.
Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro.
Cell cultures were treated with various concentrations of the peptides at different incubation intervals. Cellular morphological changes (apoptosis and necrosis) were evaluated using confocal laser scanning microscopy. The cytotoxic effects of RRM-MV on human skin cancer and normal human skin cells were quantitatively determined by cytotoxicity and cell viability assays. The effect on human erythrocytes was also determined using quantitative hemolysis assay. DNA fragmentation assay was performed to detect early apoptotic events in treated cancer cells. Furthermore, to investigate the possible cell signalling pathway targeted by the peptides treatment, the levels of p-Akt expression in skin cancer and normal cells were detected by immunoblotting.
Our results indicate that RRM-MV has a dose-dependent toxic effect on cancer cells only up to 18 h. The immunoblotting results indicated that the RRM-MV slightly increased p-Akt expression in melanoma and carcinoma cells, but did not seem to affect p-Akt expression in normal skin cells.
RRM-MV targets and lethally harms cancer cells and leaves normal cells unharmed. It is able to reduce the cancer cell viability, disrupting the LDH activity in cancer cells and can significantly affect cancer progression. Further investigation into other cell signalling pathways is needed in the process leading to the in vivo testing of this peptide to prove its safety as a possible effective treatment for skin cancer.
The Resonant Recognition Model (RRM) is a physico-mathematical model that interprets protein sequence linear information using digital signal processing methods. In this study the RRM concept was employed for structure-function analysis of myxoma virus (MV) proteins and the design of a short bioactive therapeutic peptide with MV-like antitumor/cytotoxic activity.
The analogue RRM-MV was designed by RRM as a linear 18 aa 2.3 kDa peptide. The biological activity of this computationally designed peptide analogue against cancer and normal cell lines was investigated. The cellular cytotoxicity effects were confirmed by confocal immunofluorescence microscopy, by measuring the levels of cytoplasmic lactate dehydrogenase (LDH) and by Prestoblue cell viability assay for up to 72 hours in peptide treated and non-treated cell cultures. Our results revealed that RRM-MV induced a significant dose and time-dependent cytotoxic effect on murine and human cancer cell lines. Yet, when normal murine cell lines were similarly treated with RRM-MV, no cytotoxic effects were observed. Furthermore, the non-bioactive RRM designed peptide RRM-C produced negligible cytotoxic effects on these cancer and normal cell lines when used at similar concentrations. The presence/absence of phosphorylated Akt activity in B16F0 mouse melanoma cells was assessed to indicate the possible apoptosis signalling pathway that could be affected by the peptide treatment. So far, Akt activity did not seem to be significantly affected by RRM-MV as is the case for the original viral protein.
Our findings indicate the successful application of the RRM concept to design a bioactive peptide analogue (RRM-MV) with cytotoxic effects on tumor cells only. This 2.345 kDa peptide analogue to a 49 kDa viral protein may be suitable to be developed as a potential cancer therapeutic. These results also open a new direction to the rational design of therapeutic agents for future cancer treatment.
TLRs are critical pattern recognition receptors that recognize bacterial and viral pathogen-associated molecular patterns leading to innate and adaptive immune responses. TLRs signal via homotypic interactions between their cytoplasmic Toll/IL-1R (TIR) domains and TIR domain-containing adaptor proteins. Over the course of evolution, viruses have developed various immune evasion strategies, one of which involves inhibiting TLR signaling pathways to avoid immune detection. Thus, vaccinia virus encodes the A46 protein, which binds to multiple TIR-domain containing proteins, ultimately preventing TLRs from signaling. We have identified an 11-aa-long peptide from A46 (termed viral inhibitor peptide of TLR4, or VIPER), which, when fused to a cell-penetrating delivery sequence, potently inhibits TLR4-mediated responses. VIPER was TLR4 specific, being inert toward other TLR pathways, and was active in murine and human cells and in vivo, where it inhibited LPS-induced IL-12p40 secretion. VIPER also prevented TLR4-mediated MAPK and transcription factor activation, suggesting it acted close to the TLR4 complex. Indeed, VIPER directly interacted with the TLR4 adaptor proteins MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM). Viral proteins target host proteins using evolutionary optimized binding surfaces. Thus, VIPER possibly represents a surface domain of A46 that specifically inhibits TLR4 by masking critical binding sites on Mal and TRAM. Apart from its potential therapeutic and experimental use in suppressing TLR4 function, identification of VIPER's specific binding sites on TRAM and Mal may reveal novel therapeutic target sites. Overall, we demonstrate for the first time disruption of a specific TLR signaling pathway by a short virally derived peptide.
The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, α1-proteinase inhibitor (α1PI) Pittsburgh (P1 Met → Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin.
We present here x-ray structures of free and S195A trypsin-bound α1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does
not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2′. By inference from the structure
of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction
rates of trypsin and thrombin with α1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the
antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation
to that found here for α1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between
serpins and proteinases in their Michaelis complexes.
alpha1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359-394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LPS), albeit with lower magnitude, by inducing monocyte cytokine (TNFalpha, IL-1beta) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-kappaB (NF-kappaB). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNFalpha release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit CD3/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways.
The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1' Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1' Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1' Thr increased thrombosis. Substitution of Serp-1 P2-P7 with Ala6 allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.
Apolipoprotein E (ApoE) is a lipid transport protein with expanded functions in cellular responses to tissue injury, immune regulation and cell growth. ApoE directs vascular changes that contribute to arterial protection as evidenced by the fact that isoforms of ApoE and ApoE deficiency correlate closely with accelerated plaque growth. The N-terminus of the ApoE protein has well-characterized functions, displaying lipid-binding and anti-atherogenic activity, whereas the function of the C-terminus is only partially defined. We have assessed the effects of a 14 amino acid C-terminal ApoE peptide, termed Ep1.B (239-252), on intimal neoplasia in animal models. This peptide is a fragment of a naturally processed peptide (236-252) of murine ApoE.
Ep1.B injection reduced neointimal hyperplasia after vascular surgery in rats and mice. When given during early plaque progression in ApoE-deficient mice, Ep1.B injections also prevented plaque growth. Treatment with Ep1.B did not, however, reduce established plaque growth in older mice. Peptides with alanine substitution of amino acid 249, Ep1.N, and with complete sequence reversal, Ep1.R, did not consistently inhibit plaque growth.
A naturally processed C-terminal ApoE peptide, Ep1.B, has anti-atherogenic activity indicating a role for this naturally metabolized peptide in vascular wound healing and lipid homeostasis.
α1‐Antitrypsin (α1‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α1‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure‐function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α1‐AT, specific mutants of interest, and providing an appended catalogue of the >200 α1‐AT mutants published to date. This article is protected by copyright. All rights reserved.
Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a “mouse trap.” When the RCL P1-P1′ scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the β-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.
Serpins have a wide range of functions in regulation of serine proteases in the thrombotic cascade and in immune responses, representing up to 2–10% of circulating proteins in the blood. Selected serpins also have cross-class inhibitory actions for cysteine proteases in inflammasome and apoptosis pathways. The arterial and venous systems transport blood throughout the mammalian body representing a central site for interactions between coagulation proteases and circulating blood cells (immune cells) and target tissues, a very extensive and complex interaction. While analysis of serpin functions in vitro in kinetics or gel shift assays or in tissue culture provides very necessary information on molecular mechanisms, the penultimate assessment of biological or physiological functions and efficacy for serpins as therapeutics requires study in vivo in whole animal models (some also consider cell culture to be an in vivo approach).
Biochemical fluorogenic and chromogenic assays facilitate real-time study of enzyme function. Based on the principle of enzymatic inhibition, these kinetic assays can be adapted to measure the function of serpins. Compared to traditional, electrophoretic study of serpin inhibitory complex formation, kinetic assays allow for finer temporal resolution as well as more quantitative comparisons between different conditions. This chapter describes methodology for performing real-time, kinetic measurement of serpin inhibitory activity by fluorogenic substrate conversion assay. Specifically, the methods covered include measurement of alpha-1-antitrypsin inhibitory activity against trypsin and heparin-dependent anti-thrombin III inhibitory activity against thrombin. These methods are scalable to small-volume, high-density format and can be applied for high-throughput screening of serpin activity modulators.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance, production of auto-antibodies, and inflammatory damage in multiple organs. We have tested the effect of anti-inflammatory peptide, a H2A histone fragment, termed IIIM1, on MRL/lpr mice, animal model of SLE. Oral administration of IIIM1 at early stage of disease caused reduction in proteinuria and serum anti-dsDNA antibodies. Starting the treatment at advanced stage of disease resulted in prolonged animal survival, decreased lymphadenosis and reduced levels of pathogenic or abnormal double negative CD4(-)CD8(-) cells and B220(+) cells in lymph nodes and spleen. We discovered that IIIM1 induces the production of an additional peptide, a fragment of alpha-1-antitrypsin, termed UBE. A relatively low dose (1 µg/kg) of UBE reduced proteinuria and hematuria in MRL/lpr mice. The beneficial effect of the peptide was corroborated by histological examination. Furthermore a significant reduction in serum IL17, IL12 and anti dsDNA antibodies was observed in the UBE-treated mice. Isolated CD4 cells incubated with the peptide showed a similar cytokine profile. Decreased levels of double negative CD4(-)CD8(-) and B220(+) cells were determined in lymph organs of UBE-treated animals. The beneficial effects of both UBE and IIIM1 suggest these peptides as potential drugs for SLE.
Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase. We hereby report the first structure of the stable 14-1B PAI-1 variant in its reactive center cleaved form, to a resolution of 2.0 A. The >99% complete structure represents the highest resolved structure of free cleaved PAI-1. This high-resolution structure should be of great use for drug target development and for modeling protein-protein interactions such as those of PAI-1 with vitronectin.
Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.
Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca(2+)-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037 +/- 0.007 mm2, calsequestrin: 0.042 +/- 0.021 mm2, calreticulin: 0.003 +/- 0.002 mm2, n = 46, P < .04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N + P domain: 0.038 +/- 0.012 mm2, C domain: 0.003 +/- 0.002 mm2, n = 45 rats, P < .0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N + P: 4.06 +/- 1.42, calreticulin: 0.273 +/- 0.02; n = 26, P < .009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23 +/- 2.04 seconds, calreticulin: 113.5 +/- 1.95 seconds; n = 5, P < .002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.
Analysis of in vivo Serpin functions in models of inflammatory vascular disease
- H Chen
- S Ambadapadi
- E Dai
Chen H, Ambadapadi S, Dai E et al (2018)
Analysis of in vivo Serpin functions in models
of inflammatory vascular disease. Methods Mol
Biol 1826:157-182
Viral serpin reactive center loop (RCL) peptides: design and testing
- L Zhang
- J R Yaron
- S Ambadapadi
Zhang L, Yaron JR, Ambadapadi S et al (2018)
Viral serpin reactive center loop (RCL) peptides: design and testing. Methods Mol Biol
1826:133-142
Kinetic measurement of Serpin inhibitory activity by real-time fluorogenic biochemical assay
- J R Yaron
- S Ambadapadi
- L Zhang
Yaron JR, Ambadapadi S, Zhang L et al (2018)
Kinetic measurement of Serpin inhibitory
activity by real-time fluorogenic biochemical
assay. Methods Mol Biol 1826:65-71
-1, a myxomavirus-derived immune modulating Serpin: structural design of serpin reactive center loop peptides with improved therapeutic function
- B P Mahon
- S Ambadapadi
- J R Yaron
- BP Mahon