The autoactivation of human single-chain urokinase-type plasminogen activator (uPA)

Torres-Paris, Constanza; Chen, Yueyi; Xiao, Lufan; Song, Harriet J.; Chen, Pingyu; Komives, Elizabeth A.

doi:10.1016/j.jbc.2023.105179

Cited by 2 publications

(8 citation statements)

References 65 publications

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“…HDX-MS was performed as previously described . The H 2 O buffer was PBS pH 7.4, which was lyophilized and resuspended in deuterium oxide (D 2 O) to prepare the D 2 O buffer.…”

Section: Methodsmentioning

confidence: 99%

“…HDX-MS was performed as previously described. 16 The H 2 O buffer was PBS pH 7.4, which was lyophilized and resuspended in deuterium oxide (D 2 O) to prepare the D 2 O buffer. Four μL of protein (WT 20 μM; No Light Chain 10 μM; F(−2) CT A 8.5 μM; K4 CT G 13 μM) were incubated for 5 min at 22 °C and then mixed with 56 μL of H 2 O Buffer as a control or D 2 O buffer for deuteration times of 0, 30 s, 60 s, 120 s, 600 s at the same temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Mass spectrometry was carried out using a Waters Synapt G2Si system with HDX technology (Waters Corporation) using the same methods and parameters as previously reported. 16 Peptides were identified using PLGS 2.5 (Waters Corporation), and analyzed using DynamX (Waters) and DECA as previously described. 26 Bimodal distributions of hydrogen–deuterium exchange were analyzed and deconvoluted using HX-Express3.…”

Section: Methodsmentioning

confidence: 99%

“…The expression of muPA in inclusion bodies, its refolding and its purification were carried out as described previously. , The protein concentration was measured by absorbance at 280 nm, using a Nanodrop. The extinction coefficients at 280 nm of muPA WT were 47620 M –1 cm –1 , when all cysteines were oxidized, and 46870 M –1 cm –1 when all cysteines were reduced.…”

Section: Methodsmentioning

confidence: 99%

“…In addition to the structural features, dynamic allostery also regulates the activity of serine proteases. , Hydrogen–deuterium exchange mass spectrometry (HDX-MS) has been extensively used to characterize the changes in dynamics of serine proteases in solution. − Kromann-Hansen et al suggested that the canonical chymotrypsin-like fold and this alternate fold may be in equilibrium and used HDX-MS to show that the nonchymotrypsin fold had much higher deuterium incorporation in the 170s, 180s and 220s loops . These authors were able to isolate monoclonal antibodies that stabilized the chymotrypsin-like fold, ,,, and could inhibit or stimulate the activity of muPA , suggesting that the different antibodies could shift the equilibrium between two conformational ensembles with different enzymatic activities.…”

Section: Introductionmentioning

confidence: 99%

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The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator

Torres-Paris,

Song,

Engelberger

et al. 2024

Biochemistry

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The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1CT in the disordered light chain and C122CT in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in k cat of the protease domain of muPA. In addition, hydrogen–deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the β10- and β11-strand, and the β7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the β-strands of the β-barrels to the loops on the other side of the protease domain.

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“…HDX-MS was performed as previously described . The H 2 O buffer was PBS pH 7.4, which was lyophilized and resuspended in deuterium oxide (D 2 O) to prepare the D 2 O buffer.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator

Torres-Paris,

Song,

Engelberger

et al. 2024

Biochemistry

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Urokinase-Type Plasminogen Activator Receptor (uPAR) in Inflammation and Disease: A Unique Inflammatory Pathway Activator

Hamada,

Varkoly,

Riyadh

et al. 2024

Biomedicines

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The urokinase-type plasminogen activator receptor (uPAR) is a unique protease binding receptor, now recognized as a key regulator of inflammation. Initially, uPA/uPAR was considered thrombolytic (clot-dissolving); however, recent studies have demonstrated its predominant immunomodulatory functions in inflammation and cancer. The uPA/uPAR complex has a multifaceted central role in both normal physiological and also pathological responses. uPAR is expressed as a glycophosphatidylinositol (GPI)-linked receptor interacting with vitronectin, integrins, G protein-coupled receptors, and growth factor receptors within a large lipid raft. Through protein-to-protein interactions, cell surface uPAR modulates intracellular signaling, altering cellular adhesion and migration. The uPA/uPAR also modifies extracellular activity, activating plasminogen to form plasmin, which breaks down fibrin, dissolving clots and activating matrix metalloproteinases that lyse connective tissue, allowing immune and cancer cell invasion and releasing growth factors. uPAR is now recognized as a biomarker for inflammatory diseases and cancer; uPAR and soluble uPAR fragments (suPAR) are increased in viral sepsis (COVID-19), inflammatory bowel disease, and metastasis. Here, we provide a comprehensive overview of the structure, function, and current studies examining uPAR and suPAR as diagnostic markers and therapeutic targets. Understanding uPAR is central to developing diagnostic markers and the ongoing development of antibody, small-molecule, nanogel, and virus-derived immune-modulating treatments that target uPAR.

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The autoactivation of human single-chain urokinase-type plasminogen activator (uPA)

Cited by 2 publications

References 65 publications

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator

Urokinase-Type Plasminogen Activator Receptor (uPAR) in Inflammation and Disease: A Unique Inflammatory Pathway Activator

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