The serpins (serine proteinase inhibitors) are a superfamily of proteins (350 -500 amino acids in size) that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. The serpins were last reviewed in 1994 (1). More recent studies show: 1) an expanded distribution within the kingdoms of metazoa and plantae, as well as certain viruses, 2) a surprising effect on the covalently bound target proteinase, and 3) novel biochemical and biological functions.Most serpins inhibit serine proteinases of the chymotrypsin family. However, cross-class inhibitors have been identified. The viral serpin CrmA and, to a lesser extent, PI9 (SERPINB9) inhibit the cysteine proteinase, caspase 1 (2), and SCCA1

We present a comprehensive alignment and phylogenetic analysis of the serpins, a superfamily of proteins with known members in higher animals, nematodes, insects, plants, and viruses. We analyze, compare, and classify 219 proteins representative of eight major and eight minor subfamilies, using a novel technique of consensus analysis. Patterns of sequence conservation characterize the family as a whole, with a clear relationship to the mechanism of function. Variations of these patterns within phylogenetically distinct groups can be correlated with the divergence of structure and function. The goals of this work are to provide a carefully curated alignment of serpin sequences, to describe patterns of conservation and divergence, and to derive a phylogenetic tree expressing the relationships among the members of this family. We extend earlier studies by Huber and Carrell as well as by Marshall, after whose publication the serpin family has grown functionally, taxonomically, and structurally. We used gene and protein sequence data, crystal structures, and chromosomal location where available. The results illuminate structure-function relationships in serpins, suggesting roles for conserved residues in the mechanism of conformational change. The phylogeny provides a rational evolutionary framework to classify serpins and enables identification of conserved amino acids. Patterns of conservation also provide an initial point of comparison for genes identified by the various genome projects. New homologs emerging from sequencing projects can either take their place within the current classification or, if necessary, extend it.

Expansion of “low complex” repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP—involved in important housekeeping processes—retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

Members of the serpin (serine proteinase inhibitor) superfamily have been identified in higher multicellular eukaryotes (plants and animals) and viruses but not in bacteria, archaea, or fungi. Thus, the ancestral serpin and the origin of the serpin inhibitory mechanism remain obscure. In this study we characterize 12 serpin-like sequences in the genomes of prokaryotic organisms, extending this protein family to all major branches of life. Notably, these organisms live in dramatically different environments and some are evolutionarily distantly related. A sequence-based analysis suggests that all 12 serpins are inhibitory. Despite considerable sequence divergence between the proteins, in four of the 12 sequences the region of the serpin that determines proteinase specificity is highly conserved, indicating that these inhibitors are likely to share a common target. Inhibitory serpins are typically prone to polymerization upon heating; thus, the existence of serpins in the moderate thermophilic bacterium Thermobifida fusca, the thermophilic bacterium Thermoanaerobacter tengcongensis, and the hyperthermophilic archaeon Pyrobaculum aerophilum is of particular interest. Using molecular modeling, we predict the means by which heat stability in the latter protein may be achieved without compromising inhibitory activity.

MENT (Myeloid and Erythroid NuclearTermination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Noninhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.MENT (Myeloid and Erythroid Nuclear Termination stagespecific protein), a developmentally regulated nuclear protein, is present in three main avian blood cell types (erythrocytes, lymphocytes, and granulocytes) where it is the predominant non-histone protein associated with compact heterochromatin (1). In vitro, MENT brings about a dramatic remodeling and condensation of chromatin higher order structure by forming protein "bridges" connecting separate nucleosomes in nucleosome arrays (for review see Ref.2). MENT has no homology with other known chromatin proteins but belongs to the intracellular branch of the serpin superfamily (3). Serpins were originally characterized as serine proteinase inhibitors; however, more recently certain members have been shown to be capable of inhibiting other proteinase classes such as caspases (the viral serpin crmA (4)) and papain-like cysteine proteinases (SCCA-1 (5)). The inhibitory members of the serpin family are notable for their ability to undergo a large scale conformational transition (for review see Ref. 6) that is critical for inhibition of target proteinases (7, 8) and for self-association or polymerization (9 -11). Multiple sequence alignments and comparison with known serpin structures reveal that MENT contains a large insertion, the "M-loop," between the C-and D-helices. This loop contains two critical functional motifs as follows: a classical nuclear localization signal (NLS) 1 that is required for nuclear import, and an AT-hook motif that is involved in chromatin and DNA binding (3). Like other serpins, MENT possesses a reactive center loop (RCL) 2 through which interaction with a cognate proteinase occurs. The presence of an inhibitory...

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 nonisomorphous 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 ␤-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 ␣-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 ␣-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.Maspin (mammary serine proteinase inhibitor (SERPINB5)) was initially identified as a tumor-suppressing serpin downregulated in invasive mammary carcinoma cell lines (1). Maspin loss in numerous cancers (including breast, prostate, squamous cell carcinoma, gastric cancer, and lung) correlates with metastasis and a poor clinical prognosis (for a review, see Ref.2). In contrast, high levels of maspin expression in certain cancers (in particular, pancreatic and ovarian cancer) correlate with tumor invasion and poor survival. Like other clade B serpins (3), maspin has a nucleocytoplasmic distribution, however it is also found at the cell surface (1, 4, 5). The intracellular role of maspin is at present unclear, but it has been suggested to play a role in apoptosis pathways (6). A large body of evidence suggests that maspin has an important extracellular role: it can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro (1,7,8). Maspin also plays a fundamental role in early embryonic development; murine knock-out studies reveal that it is essential for proper organization of the epiblast (9). Consistent with a complex role in tumorigenesis, maspin also exhibits anti-angiogenic activity (10), and expression of the maspin gene has been demonstrated to be under the control of the oncogenic transcription factors p53 and p63 (11,12). Because the failure to properly control proteolytic activity can result in disruption of the basement membrane and promote tumor invasion, it was initially hypothesized that maspin may exert its anti-metastatic effect by functioning as a pericellular protease inhibitor (1).Serpins are unusual ...

We present a comprehensive alignment and phylogenetic analysis of the serpins, a superfamily of proteins with known members in higher animals, nematodes, insects, plants, and viruses. We analyze, compare, and classify 219 proteins representative of eight major and eight minor subfamilies, using a novel technique of consensus analysis. Patterns of sequence conservation characterize the family as a whole, with a clear relationship to the mechanism of function. Variations of these patterns within phylogenetically distinct groups can be correlated with the divergence of structure and function. The goals of this work are to provide a carefully curated alignment of serpin sequences, to describe patterns of conservation and divergence, and to derive a phylogenetic tree expressing the relationships among the members of this family. We extend earlier studies by Huber and Carrell as well as by Marshall, after whose publication the serpin family has grown functionally, taxonomically, and structurally. We used gene and protein sequence data, crystal structures, and chromosomal location where available. The results illuminate structure–function relationships in serpins, suggesting roles for conserved residues in the mechanism of conformational change. The phylogeny provides a rational evolutionary framework to classify serpins and enables identification of conserved amino acids. Patterns of conservation also provide an initial point of comparison for genes identified by the various genome projects. New homologs emerging from sequencing projects can either take their place within the current classification or, if necessary, extend it.