The crystal structure of the catalytic domain of human urokinase-type plasminogen activator

In striking contrast to most other members of the chymotrypsin family of serine proteases, tissue-type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. The zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 5-10 for t-PA. This exceptional property of t-PA, however, is not shared by urokinase (u-PA), a plasminogen activator that is very closely related to t-PA. The molecular basis of this important functional distinction between these two intimately related serine proteases has not been previously investigated. Based on observation of the recently described structures of the protease domains of two-chain t-PA and u-PA, and molecular modeling of the corresponding single-chain enzymes, we propose that the presence or absence of an acidic residue at position 144 (chymotrypsin numbering system) is the primary determinant of the distinct zymogenicities of the two enzymes. Consistent with this hypothesis, mutation of histidine 144 of t-PA to an acidic residue, as in u-PA, selectively suppressed the activity of single-chain t-PA and thereby significantly enhanced the enzyme's zymogenicity. A variant of t-PA containing an aspartate residue at position 144, for example, exhibited a zymogenicity of 150, compared to a value of 9 for wild type t-PA and 250 for u-PA.Many critical biological processes depend on specific cleavage of individual target proteins by serine proteases (1-3). One important example is the dissolution of blood clots in which the initiating and rate-limiting step is activation of the circulating zymogen plasminogen (4, 5). In mammalian systems, activation of plasminogen is accomplished by two closely related enzymes, tissue-type plasminogen activator (t-PA) 1 and urokinase (u-PA) (4 -7). t-PA and u-PA possess an extremely high degree of structural similarity (8, 9), share the same primary endogenous substrate and inhibitors (4), and exhibit remarkably stringent substrate specificity (5). In spite of these striking similarities, however, there are clear functional distinctions between the two enzymes. One particularly intriguing distinction is that, by contrast to single-chain u-PA, single-chain t-PA possesses unusually high catalytic activity and is therefore not a true zymogen (10 -13).Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 10 4 to 10 6 (14, 15). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (16), and the catalytic efficiency of ur...

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confidence: 97%

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confidence: 99%

Converting Tissue-type Plasminogen Activator into a Zymogen

Tachias

Madison

1996

“…In this report we extended this approach to exploit the localized activity of the uPA protease on tumor cells. uPA and tPA possess an extremely high degree of structural similarity (56,57), share the same primary physiological substrate (plasminogen) and inhibitors (PAI-1 and PAI-2) (58), and exhibit restricted substrate specificity. Recent elegant genetic studies using substrate phage display and substrate subtraction phage display identified peptide substrates that are cleaved with high efficiency as well as high selectivity by either uPA or tPA (59,60).…”

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confidence: 99%

Targeting of Tumor Cells by Cell Surface Urokinase Plasminogen Activator-dependent Anthrax Toxin

Liu

Bugge

Leppla

2001

161

169

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.

“…These two proteases possess extremely high structural similarity (7,8), share the same primary physiological substrate (plasminogen) and inhibitor (plasminogen activator inhibitor, type 1) (3), and, unlike plasmin, exhibit remarkably stringent substrate specificity (9 -11). Despite their striking similarities, the physiological roles of t-PA and u-PA are distinct (5,6), and many studies (5,6,(12)(13)(14)(15)(16)(17)(18) suggest selective inhibition of either enzyme might have beneficial therapeutic effects.…”

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confidence: 99%

Optimal Subsite Occupancy and Design of a Selective Inhibitor of Urokinase

Ke¹,

Coombs²,

Tachias³

et al. 1997

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify peptides that are cleaved 840 -5300 times more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2, represents optimal subsite occupancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.