The serpins are an expanding superfamily of structurally similar but functionally diverse proteins anoxic brain injury recovery

• gary A. Silverman toa fnb,

• phillip I. Bird toc,

• robin W. Carrell tod,

• frank C. Church toe,

• paul B. Coughlin tof,

• peter G. W. Gettins tog,

• james A irving toc,

• david A. Lomas tod,

• cliff J. Luke toa,

• richard W. Moyer toh,

• philip A. Pemberton i,

• eileen remold-O’donnell toj,

• guy S. Salvesen tok,

• james travis tol and

• james C. Whisstock toc

• from the toadepartment of pediatrics, division of newborn medicine, children’s hospital, harvard medical school, boston, massachusetts

02115, tocdepartment of biochemistry and molecular biology, monash university, melbourne, victoria 3800, australia, toddepartments of haematology and medicine, university of cambridge, wellcome trust centre for molecular mechanisms in disease,

anoxic brain injury recovery

Cambridge institute for medical research, wellcome trust/MRC building, hills road, cambridge CB2 2XY, united kingdom, toedivision of hematology-oncology/medicine, university of north carolina, chapel hill, north carolina 27599, tofdepartment of medicine, box hill hospital, monash university, melbourne, victoria 3128, australia, togdepartment of biochemistry and molecular biology, university of illinois, chicago, illinois 60612, tohdepartment of molecular genetics and microbiology, university of florida college of medicine, gainesville, florida 32610,

IArriva pharmaceuticals, alameda, california 94501, tojcenter for blood research, harvard medical school, boston, massachusetts 02115, tokburnham institute, san diego, california 92037, toldepartment of biochemistry and molecular biology, university of georgia, athens, georgia 30602

anoxic brain injury recovery

The serpins ( serine proteinase inhibitor s) 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 1 (SERPINB3) neutralizes the potent papain-like cysteine proteinases, cathepsins L, K, and S ( 3).Anoxic brain injury recovery in addition, several members no longer function as proteinase inhibitors but perform other roles such as hormone transport

(thyroid-binding globulin (SERPINA6), corticosteroid-binding globulin (SERPINA7)), and blood pressure regulation (angiotensinogen

(SERPINA8)) ( 1).

Data base searching provides evidence for ∼500 serpins, with full-length coding sequences known or predicted for about one-half

Of those ( 4). A phylogenetic analysis divides serpins into 16 clades (see supplemental data, table A) and 10 highly diverged “orphans”

( 4). These data facilitate the construction of a consistent expandable nomenclature (see supplemental data for serpin nomenclature

Guidelines, table B).

The completed DNA sequences of several organisms have yielded insight into the complexity of the family.Anoxic brain injury recovery the caenorhabditis elegans, drosophila melanogaster, and arabidopsis thalianagenomes encode for ∼20,000, 13,000, and 25,000 genes, respectively. However, these three species harbor ∼9, 32, and 13 serpin

Genes, respectively. The nonlinear relationship among the number of serpin genes, relative to the total gene number, suggests

That at least a subset of serpins has evolved divergent functions despite a striking degree of sequence and structural conservation.

Serpin conformations

Serpins adopt a metastable conformation that is required for their inhibitory activity ( 5). This conformation consists of a conserved secondary structure comprised of β-sheets A, B, and C and at least 7 α-helices

(most typically have 9, lettered A– I; fig. 1 A).Anoxic brain injury recovery the RSL, which contains the proteinase recognition site, is an exposed, flexible stretch of ∼17 residues tethered between

Β-sheets A and C. Serpins can undergo major structural rearrangements that involve alternative conformations for the RSL,

Β-sheet A, and the attached strand 1 of β-sheet C. Considering only intramolecular structural changes, serpins can convert

To the more stable latent form (fig. 1 B). The RSL inserts into the middle of β-sheet A to give a fully antiparallel β-sheet, and s1c is extracted from β-sheet C

To provide an exposed “return” from the bottom of the serpin. Serpins in the latent conformation are noninhibitory but can

Be converted back to the active state by denaturation and refolding.Anoxic brain injury recovery the T m for unfolding of latent PAI1 (SERPINE1) is 17 °C higher than that for the native state (reviewed in ref. 6). The most stable state for inhibitory serpins is the RSL-cleaved form, in which the RSL has fully inserted into β-sheet

A, as in the latent conformation, but without the need to extract s1c from β-sheet C (fig. 1 C). Estimates of the T m for unfolding of such conformations are 120 °C, compared with ∼60 °C for the native state ( 7).

Serpin inhibitory mechanism

Serpins inhibit serine proteinases by an irreversible suicide substrate mechanism when the interaction proceeds down the inhibitory

Arm of a branched pathway (fig. 2) ( 6). In the inhibitory pathway, the proteinase initially forms a noncovalent michaelis-like complex (fig. 1 D) through interactions with residues flanking the scissile bond (P1–P1′).Anoxic brain injury recovery attack of the active site serine on the scissile

Bond leads to a covalent ester linkage between ser-195 of the proteinase and the backbone carbonyl of the P1 residue and cleavage

Of the peptide bond ( 6). It is likely that only at this stage, with removal of the restraint, does the RSL start to insert into β-sheet A and transport

The covalently bound proteinase with it. Upon complete loop insertion the proteinase is translocated by over 70 Å, and its

Active site is distorted (fig. 1 E). The alignment of the active site catalytic triad is altered by as much as 3 Å, and the P1 side chain is removed from the

S1 pocket. Also, 40% of the body of the proteinase shows no traceable electron density. Proteinase distortion and hence inactivation

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Results from compression of the proteinase against the base of the serpin as a consequence of the inserted RSL being just

The right length. The energy needed to effect the distortion may come from the much greater stability of the cleaved loop-inserted

Conformation compared with the native-like conformation. The net result of this conformational rearrangement is kinetic trapping

Of the acyl intermediate due to slowing of the deacylation steps of the normal substrate reaction by 6–8 orders of magnitude

( k 5 in fig. 2). Because of the small values for k 5 (complex t ≅ hours to weeks), serpin-proteinase complexes in vivowould bind to their receptors and be cleared (complex t ≅ minutes) long before significant complex decay could occur.Anoxic brain injury recovery

The point in transit where the enzyme activity is reduced sufficiently to commit the intermediate to the kinetic trap is not

Known but in part contributes to the branched nature of the pathway and the ultimate fate of the complex. If, for example,

RSL movement is impeded, the enzyme may successfully complete the deacylation step and escape before it is irreversibly trapped.

This noninhibitory pathway yields an active proteinase and a cleaved, inactive serpin. The ratio of serpin products (complex versuscleaved) thus reflects a competition between the rate of ester hydrolysis ( k 3 in fig. 2) and that of loop insertion ( k 4 in fig. 2) to the point of proteinase distortion. This ratio is signified also by the stoichiometry of inhibition, which is defined

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As ( k 3 + k 4)/ k 4, i.E.The number of moles of serpin needed to inhibit 1 mol of proteinase as a kinetically trapped complex.

This mechanism accounts for the requirements for effective inhibition by serpins, which include a critical RSL length, appropriate

Residues within the loop that are compatible with rapid and favorable burial into β-sheet A, and the presence of ser in the proteinase active site ( 6). Such a mechanism is adaptable to the inhibition of cysteine proteinases by serpins, with the difference being that the

Kinetically trapped intermediate is a thiol ester rather than an oxy ester. The detection of crma, a serpin that inhibits

Cysteine proteinases of the caspase family, in the loop-inserted cleaved conformation supports the feasibility of a common

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Inhibitory mechanism ( 9), whereas the detection of an SDS-stable complex between SCCA1 and cathepsin S (a cysteine proteinase of the papain family)

Provides evidence for the formation of a stable, covalent thiol ester-type linkage ( 3). The few convincing reports of reversible inhibition, such as of single-chain upa by PCI (SERPINA5) ( 10) or of chymotrypsin by α 2AP (SERPINF2) ( 11) may represent special cases in which unusual stabilization of the initial noncovalent michaelis-like complex blocks progression

To the substrate reaction.

Ov-serpins (B clade)

In 1993 amino acid similarities among chicken ovalbumin (ov), PAI2 (SERPINB2), and MNEI (SERPINB1) led to the identification

Of a subgroup of the serpin superfamily ( 13).Anoxic brain injury recovery the N and C termini of the ov-serpins are shorter than the prototypical serpin α 1AT, and they also lack a classical secretory signal peptide. At present, there are 13 human ov-serpins (see supplemental data,

Table B). They map to 6p25 and 18q21 and fall into two classes based on a single difference in gene structure ( 14). Like ovalbumin, many of the 18q21 serpin genes have an exon encoding a polypeptide loop between helices C and D (CD loop)

That may contribute to accessory functions.

Unlike ovalbumin itself, most ov-serpins reside intracellularly with a cytoplasmic or nucleocytoplasmic distribution. However,

Several ov-serpins (PAI2, megsin (SERPINB7), MNEI, maspin (SERPINB5), and the sccas (SERPINB3 and -4)) may function extracellularly

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As they are released from cells under certain conditions. Release may be facilitated by an embedded, noncleaved hydrophobic

N-terminal signal sequence and appears to involve both conventional and non-endoplasmic reticulum-golgi secretory pathways

( 15). Regardless of how ov-serpins are released from cells, those with RSL cysteine or methionine residues are susceptible to

Oxidative inactivation and are likely to have a limited half-life in the extracellular milieu.

With the possible exception of maspin, all human ov-serpins are functional, competitive inhibitors of serine or cysteine proteinases.

Several members of the group inhibit more than one proteinase, and dual reactive sites (utilization of more than one P1 residue)

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Have been described for PI6 (SERPINB6), PI8 (SERPINB8), PI9, SCCA1, SCCA2, and MNEI (for example see ref. 16). However, the CD loops of the ov-serpins have the potential to interact with other proteins. For example, the CD loop of

PAI2 is required for its cell survival function ( 17) and is a target for transglutamination ( 18). Bomapin (SERPINB10; like the chicken ov-serpin, MENT, see below) carries a nuclear localization signal in its CD loop that

Presumably interacts with a nuclear importin ( 19).

The physiological functions of ov-serpins are still emerging. PAI2 may play a role in the regulation of extracellular matrix

Remodeling through the inhibition of upa, as high PAI2 and low upa levels correlate with a positive prognosis in breast cancer

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( 20). Also, PAI2 may have a structural role inside some cells (perhaps keratinocytes) as suggested by its ability to spontaneously

Polymerize and undergo transglutamination ( 21).

Many ov-serpins reside in proteinase-secreting cells ( 22). For example, PI9, a potent inhibitor of granzyme B, is also present in cytotoxic lymphocytes. Because PI9 can protect cells

Against granzyme B-mediated apoptosis, it probably protects cytotoxic lymphocytes from autodestruction due to misdirected

Granzyme B. A similar cytoprotective role can be envisaged for PI6, PI8, MNEI, PAI2, and the sccas. In addition, endogenous

Or exogenous ov-serpins may protect bystander cells and tissue from proteolytic damage. Studies in rats show that recombinant

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MNEI delivered to the airways prevents lung injury by neutrophil proteinases and point to its potential in treating inflammatory

Lung disease ( 23).

The ability of many ov-serpins to inhibit more than one proteinase and their presence in epithelial cells suggest that they

Play a role in barrier function or host defense against microbial or viral proteinases. For example, PI9 inhibits bacillussubtilisin, and PI8 inhibits furin, a subtilisin-related enzyme ( 24, 25). Additional functions of ov-serpins include the regulation of: 1) cell growth or differentiation, as exemplified by the

Role of megsin in megakaryocyte differentiation ( 26), 2) tumor cell invasiveness and motility, as shown by the inhibitory role of maspin in breast and prostate tumors ( 27), and 3) angiogenesis (see below).Anoxic brain injury recovery

Pigment epithelium-derived factor and other serpins that may interfere with angiogenesis

PEDF (SERPINF1) is a secreted, noninhibitory serpin that was isolated from retinal pigment epithelial cells but is also detected

In liver, lung, heart, spleen, brain, and testis ( 6). This factor promotes the survival and differentiation of retinal photoreceptors, cerebellar granule neurons, and spinal

Motor neurons. Dawson et al. ( 33) show that PEDF inhibits neovascularization of the rat cornea and endothelial cell migration in vitro. In the cell migration assay, PEDF was as potent as other angiogenesis inhibitors such as angiostatin, endostatin, and thrombospondin-1.

Moreover, PEDF antagonized the effects of the angiogenesis inducers, VEGF, basic fibroblast growth factor, platelet-derived

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Growth factor, and interleukin 8. PEDF expression in the eye increases with rising oxygen tension (just the opposite of VEGF).

Thus, VEGF and PEDF appear to counter-regulate blood vessel growth in the eye by enhancing and antagonizing angiogenesis during

Hypoxic and hyperoxic conditions, respectively.

PAI1, maspin, and RSL-cleaved ATIII (SERPINC1) have been shown to interfere with angiogenesis in various assay systems ( 34-36). However, it has yet to be determined whether any of these molecules are truly involved in the physiologic or pathologic

Regulation of blood vessel growth.

Viral serpins

Serpins are found within a number of genera within the subfamilies of the vertebrate poxviruses and the gammaherpesviruses

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(see supplemental data, table D). To date, none of the serpins are required for virus growth in cell culture. Within the vertebrate

Poxvirus genera, each orthopoxvirus (variola, vaccinia, and rabbitpox) encodes three highly conserved serpins, SPI-1, SPI-2/crma, and SPI-3. Each targets different

Types of proteinases (table D). The prototypic member of the leporipoxvirus genus (myxoma virus) also encodes three serpins, SERP1–3. Within the avipoxvirus genus, the fowlpox virus genome contains 5 serpin genes (table D). Other vertebrate poxviruses (molluscum contagiosum and

ORF viruses) lack serpin genes. All genera of poxviruses encode serpins with putative asp P1 residues, and the leporipoxviruses, orthopoxviruses, and fowlpox viruses all have a member with a putative arg P1 residue (table D).Anoxic brain injury recovery only the orthopoxviruses have a serpin with a phe at the putative P1 site. For more information on orthopoxvirus and leporipoxvirus serpins see supplemental data.