Serpins

Brief Overview of Serpins (Serine Protease Inhibitors)

Overview of the Research in the Laboratory: Our research emphasis over the past 20 years has been focused on serpins (serine protease inhibitors), in particular heparin cofactor II (HCII) compared to antithrombin (AT) and protein C inhibitor (PCI, also named plasminogen activator inhibitor-3) and a1-protease inhibitor (a1-PI), and recently, we have been doing a lot of things with plasminogen activator inhibitor-1 (PAI-1). Our science is to better understand the pathophysiology and structure-activity relationships of heparin-binding serpins, thus, we perform the following types of studies:

•Structure-Activity Relationships (Protein Biochemistry and Biological Chemistry)
•Mechanisms of Disease (Cell and Molecular Biology)
•Pathobiology of Thrombosis and Cancer
•"Biostructural Pathology" (as coined by Robin Carrell a few years ago)

Physiological Roles of Serpins (serine protease inhibitors). Serpins in General: The primary function of serpins is to regulate the proteolytic activity of serine (and some cysteine) proteases that are involved in such processes as coagulation, fibrinolysis, complement activation, inflammation, tumor metastasis, and extracellular matrix remodeling. However, some have functions that do not involve protease inhibition, such as hormone transport [cortisol-binding protein and thyroxine binding globulin] or blood pressure regulation [angiotensinogen]. The majority of known serpins are secreted proteins, but several, such as plasminogen activator inhibitor-2 (PAI-2) and protease inhibitor-6, are intracellular serpins.

Certain genetic disorders related to deficiencies in serpin concentration or function have paved the way for understanding the physiological role of a number of serpins. For example, congenital antithrombin (AT) deficiency is associated with an increased risk for thrombotic disorders. AT deficiency has been found in 2.4% of young patients with venous thrombosis and cross-sectional studies have shown that between 30 and 80% of carriers will have a thrombotic episode in early adulthood. These clinical findings have enabled us to appreciate the physiological importance of AT as an anticoagulant serpin. The development of emphysema is related to a deficiency in a1-protease inhibitor (a1-PI). a1-PI is thought to protect tissues from released proteoytic enzymes, particularly at the periphery of a site of inflammation. In the lung, a1-PI inhibits elastase released from activated neutrophils in the lower respiratory tract; thus, deficiencies in a1-PI lead to progressive proteolytic destruction of alveoli. Deficiencies in C1-inhibitor, which inhibits complement components and proteases of the intrinsic coagulation pathway, lead to hereditary angioedema. Recently, serpins have also been implicated in tumor metastasis. The non-inhibitory serpin, maspin, is thought to be a tumor suppressor that may play a role in human breast cancer. At least one serpin from an infectious organism has been shown to attenuate the host inflammatory response. CrmA, a cow pox virus serpin, inhibits interleukin-1b converting enzyme, which converts the pro-inflammatory cytokine, interleukin-1b, to its active form and granzyme B, which is used by cytolytic cells to cause apoptosis in target cells.

Serpin Structure and Mechanism of Proteinase Inhibition: Serpins are a superfamily of single chain proteins of approximately 350 - 450 amino acids whose prototype is a1-PI. Over 400 different serpins have been identified in organisms including viruses, plants, insects, animals, and even procaryotes, and from a variety of locations from plasma to neuroglia. Plasma serpins are variably glycosylated, but the carbohydrate side chains are not necessary for activity. Serpins contain a reactive center on an exposed peptide loop that is recognized as a substrate by the protease. Serpins have a highly mobile reactive site loop that can change conformation in response to various stimuli, such as the heparin-induced conformational change in the reactive site loop of antithrombin (AT).

Three-dimensional Structure: Serpins have been studied extensively using biochemical, molecular biology, and crystallographic approaches and a very detailed picture has emerged regarding their three-dimensional structure. Crystal structures have been obtained of native uncleaved serpins, a non-inhibitory serpin (ovalbumin), reactive site loop-cleaved serpins, encounter complexes between serpin-target proteases, and even covalent serpin-protease complexes. Serpins have a highly ordered, ellipsoidal tertiary structure which consists of nine a-helices and three ß-sheets arranged in a stressed configuration. From the central ß-sheet A, strand 5 emerges to become the reactive site loop approximately 15 residues upstream (P15) of the reactive site bond and then re-enters the body of the protein as strand 1 of ß-sheet C. The reactive site bond, termed the P1-P1' bond by the nomenclature of Schechter and Berger, is positioned such that it is readily accessible to proteases. In cleaved serpins, the two residues of the P1-P1' bond are fixed on opposite sides of the protein and separated by 70 Å. Residues P16 to P1 of the reactive site loop form a central strand (4A) of the A ß-sheet with the P1 residue located at the opposite end of its original location in the protein. Residues downstream from the scissile bond move to the surface of the protein within a second ß-sheet. The conformational change that occurs during cleavage is detected biochemically as a transformation from a labile to a heat-stable form.

Another unique feature of serpin structure has been determined by comparing the crystal structures of ovalbumin and the active molecule of dimeric AT. The reactive site loop of the intact molecule of dimeric thrombin is partially inserted at the P16 and P15 residues, the so-called "hinge region", into the A ß-sheet and forms hydrogen-bonding interactions with strands 3A and 5A. The P9-P15 sequence is highly conserved in all inhibitory serpins and mutations in this region result in loss of inhibitory activity. Furthermore, the amino acids of the hinge region have small side chains, which allows loop flexibility necessary for complex formation. In contrast, the "reactive site loop" of ovalbumin is in a fully extended, rigid alpha-helical conformation that is unable to conform to the active site of a protease, which explains its lack of inhibitory activity. Thus, partial loop insertion serves an important purpose in the activity of inhibitory serpins, and it is thought that significant loop insertion occurs with the formation of the serpin-protease complex. The driving force for this conformational change is thought to be the energy loss associated with the increased loop insertion in the complexed serpin.

Structural Features of Heparin-binding Serpins: A sub-family of serpins exists whose inhibitory activity is greatly accelerated upon binding of heparin and other negatively charged polyanions, such as heparan sulfate and dermatan sulfate. The members of this group include heparin cofactor II (HCII), antithrombin (AT), protein C inhibitor (PCI, also named plasminogen activator inhibitor-3), protease nexin 1 (PN-1), and plasminogen activator inhibitor-1 (PAI-1). Heparin is a highly negatively charged glycosaminoglycan consisting of alternating glucosamine and iduronic acid monomers. Commercial heparin is prepared from porcine and bovine tissues, and is present as a heterogeneous mixture of fragments that range from 5000 to 30,000 Kd. Dermatan sulfate contains alternating D-glucoronic or L-iduronic acid and N-acetyl-D-galactosamine monomers. Heparin has a more anionic character due to a higher degree of sulfation when compared to heparan sulfate and to dermatan sulfate.