Activation of the Zymogen to Urokinase-Type Plasminogen Activator Is Associated with Increased Interdomain Flexibility

Behrens, Manja A.; Bøtkjær, Kenneth A.; Goswami, Sumit; Oliveira, Cristiano L. P.; Jensen, Jan K.; Schar, Christine R.; Declerck, Paul; Peterson, Cynthia B.; Andreasen, Peter A.; Pedersen, Jan Skov

doi:10.1016/j.jmb.2011.05.026

Cited by 13 publications

(15 citation statements)

References 45 publications

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“…To examine the mechanism of spontaneous Z-a1AT polymerization under physiological conditions, we employed small-angle x-ray scattering (SAXS), a solutionbased structural technique in which the solution conditions can easily be varied. Whereas crystal structures provide static atomic-resolution models, SAXS can give the overall shape and thus produce low-resolution molecular models of proteins in solution (43). From our study, we conclude that the Z-a1AT trimer in solution is indeed similar to the one observed in the protein crystal.…”

Section: Biophysical Journal 107(8) 1905-1912supporting

confidence: 56%

The Shapes of Z- α 1 -Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

Behrens

Sendall

Pedersen

et al. 2014

Biophysical Journal

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Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor a1antitrypsin (a1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-a1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-a1AT polymers in solution. The data show that the Z-a1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.

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Section: Biophysical Journal 107(8) 1905-1912supporting

confidence: 56%

The Shapes of Z- α 1 -Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

Behrens

Sendall

Pedersen

et al. 2014

Biophysical Journal

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“…Similarly, an aptamer against an IgG 1 Fc-fragment with molecular mass of 8 kDa had maximum length of about 45 Å, while the IgG 1 Fc-fragment has a molecular weight of 50 kDa and a maximum length of approximately 70 Å [44]. With a molecular mass for upanap-126 of about 26 kDa, its length may well approach 100 Å, which should be sufficient to cover a region from the primary binding site in the uPA SPD up to its EGF-domain [45]. The observation that upanap-126 inhibits the binding of pro-uPA to uPAR with a lower IC 50 than the binding of active two-chain uPA to uPAR (Figure 3A) could be caused by a slightly differential affinity of upanap-126 for pro-uPA versus active uPA or differences in the interaction of pro-uPA and active uPA with uPAR.…”

Section: Discussionmentioning

confidence: 99%

“…The observation that upanap-126 inhibits the binding of pro-uPA to uPAR with a lower IC 50 than the binding of active two-chain uPA to uPAR (Figure 3A) could be caused by a slightly differential affinity of upanap-126 for pro-uPA versus active uPA or differences in the interaction of pro-uPA and active uPA with uPAR. Previously, we showed that uPA acquire an increased overall interdomain flexibility upon conversion of pro-uPA to active uPA [45]. …”

Section: Discussionmentioning

confidence: 99%

Targeting Tumor Cell Invasion and Dissemination In Vivo by an Aptamer That Inhibits Urokinase-type Plasminogen Activator through a Novel Multifunctional Mechanism

Bøtkjær

Deryugina

Dupont

et al. 2012

Molecular Cancer Research

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Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic since the topology of the proteases’ active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2′-fluoro-pyrimidine RNA molecules using as bait a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains. One pro-uPA binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalysed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complexes both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumour cell invasion in vitro, in the Matrigel assay, and in vivo, in the chick embryo assay of cell escape from microtumours. Finally, upanap-126 significantly reduced the levels of tumour cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings demonstrate that utilisation of upanap-126 represents a novel multi-functional mechanistic modality for inhibition of uPA-dependent processes involved in tumour cell spread.

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“…SAXS analysis was already applied to characterize the overall shape of full-length pro-uPA and active uPA [ 25 ]. In order to obtain low-resolution structural information regarding the relative position of the aptamer in the quaternary complex with uPA, we performed SAXS analysis of upanap-12 alone and in complex with pro-uPA.…”

Section: Resultsmentioning

confidence: 99%

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

et al. 2015

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Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

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Activation of the Zymogen to Urokinase-Type Plasminogen Activator Is Associated with Increased Interdomain Flexibility

Cited by 13 publications

References 45 publications

The Shapes of Z- α 1 -Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

The Shapes of Z- α 1 -Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

Targeting Tumor Cell Invasion and Dissemination In Vivo by an Aptamer That Inhibits Urokinase-type Plasminogen Activator through a Novel Multifunctional Mechanism

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

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