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Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent ( K i = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Background: The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined., Results: We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities., Conclusions: In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Members of the serpin (serine protease inhibitor) superfamily fold into a metastable conformation that is crucial for proper function. As a consequence, serpins are susceptible to mutations that cause misfolding and the intracellular accumulation of pathogenic polymers. The mechanism of serpin polymerisation remains to be resolved, however, over the past two decades the 'loop-sheet' hypothesis has gained wide acceptance. In this mechanism the reactive centre loop of one serpin monomer inserts into the beta-sheet A of another (in trans), in a manner similar to what is seen for reactive centre loop-cleaved and latent conformations (in cis). The hypothesis has been refined in response to certain experimental data, but it has proved difficult to assess the various propositions without creating molecular models. Here we evaluate the loop-sheet mechanism by creating models of pentamers of the archetypal serpin alpha(1)-antitrypsin. We conclude that an inescapable consequence of the loop-sheet mechanism is polymer compaction and rigidity, properties that are inconsistent with the 'beads-on-a-string' morphology of polymers obtained from human tissue. The recent crystal structure of a domain-swapped serpin dimer suggests an alternative mechanism that is consistent with known polymer properties, including the requirement of partial unfolding to induce polymer formation in vitro, and polymerisation from a folding intermediate in vivo.

Maspin (SERPINB5) is a tumor suppressor lost in breast and prostate cancer whose molecular function is unknown. It is a non-inhibitory member of the clade B serpins suggested to play a role in a plethora of intracellular and extracellular settings, yet its normal cellular distribution has never been clarified. Here we investigate the distribution of maspin in non-transformed human epithelial cells. By indirect immunofluorescence, maspin has a nucleocytoplasmic distribution in breast (MCF10A) and prostate (RWPE-1) cells and, by immunoblotting and pulse-chase analyses, is neither glycosylated nor secreted. Cell surface biotinylation studies also show that maspin is not present at the cell surface. Differentiation of MCF10A cells into three-dimensional acini results in the redistribution of maspin from the nucleus to the cytoplasm but does not result in secretion. Addition of an efficient conventional signal peptide to maspin directs it into the secretory pathway and results in glycosylation but not secretion. We further show that maspin in the cytoplasm of MCF10A cells is a soluble monomeric protein that is not detectably associated with the cytoskeleton or other extractable components. Taken together, these results suggest that maspin is restricted to an intracellular, possibly nuclear, role in which it influences cell-matrix interactions indirectly. It is probably released only as a consequence of cell damage or necrosis.

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.

Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.

Antithrombin is a serine proteinase inhibitor (serpin) which controls the process of coagulation. It has a well defined structure, consisting of three beta-sheets, nine alpha-helices and a reactive centre loop (RCL). The RCL contains the reactive centre which harbours a bait sequence for target proteases; cleavage results in inhibition by a unique mechanism. The inhibitory activity of antithrombin is controlled by its interaction with the co-factor, heparin, which accelerates its interaction with target proteases. This ensures that heparin and its newer derivatives, such as heparin pentasaccharide, are the mainstay therapeutics for control of thrombosis or inappropriate clotting. The clinical importance of antithrombin is manifested by its clear association with thrombosis when deficiency states occur.

Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C. The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment.

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia). [ABSTRACT FROM AUTHOR]

The article discusses that polymers of serpin proteins underlie diseases, including neurodegenerative disorders. It is said that Alois Alzheimer has offered a description on the abnormal aggregates of the amyloid protein in the brain of a patient who has died of dementia. The known conformational diseases include prion diseases, Parkinson's disease, Huntington's disease and several disorders known as serpinopathies. It is mentioned that the serpin polymer is correctly folded and resembles the relaxed serpin, which does not evoke protective signalling pathway.

Maspin (SERPINB5) is a member of the Clade B subgroup of the large superfamily of serine protease inhibitors. It is proposed that maspin is a tumor suppressor; however, its molecular role remains to be elucidated. Here we report the characterization of a mouse monoclonal antibody directed against human maspin. This antibody, 16F7, recognizes maspin in both its native and denatured form, unlike several other commercial antibodies tested in this study. It will be a useful and versatile tool for future analyses of the biological function of maspin. [ABSTRACT FROM AUTHOR]

Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin super family, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure ofa murine antichymotrypsin orthologue to 2.1 Å, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A β-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90° to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 ± 0.9 ⊗ 106 M-1 s-1) and cathepsin G (ka: 7.9 ± 0.9 ⊗ 105 M-1 s-1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4–P6′ region of the RCL, the distal hinge, and the loop between strands 4B and 5B. [ABSTRACT FROM AUTHOR]

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-Å resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A Β-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E α-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G α-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor. [ABSTRACT FROM AUTHOR]