Modulation of RNA-binding properties of the RNA helicase UPF1 by its activator UPF2.

The NMD helicase UPF1 is a prototype of the superfamily 1 (SF1) of RNA helicases that bind RNA with high affinity and translocate on it in an ATP-dependent manner. Previous studies showed that UPF1 has a low basal catalytic activity that is greatly enhanced upon binding of its interaction partner, UPF2. Activation of UPF1 by UPF2 entails a large conformational change that switches the helicase from an RNA-clamping mode to an RNA-unwinding mode. The ability of UPF1 to bind RNA was expected to be unaffected by this activation mechanism. Here we show, using a combination of biochemical and biophysical methods, that binding of UPF2 to UPF1 drastically reduces the affinity of UPF1 for RNA, leading to a release of the bound RNA. Although UPF2 is capable of binding RNA in vitro, our results suggest that dissociation of the UPF1-RNA complex is not a consequence of direct competition in RNA binding but rather an allosteric effect that is likely mediated by the conformational change in UPF1 that is induced upon binding its activator. We discuss these results in light of transient interactions forged during mRNP assembly, particularly in the UPF1-dependent mRNA decay pathways.

Structural study of the uPA-nafamostat complex reveals a covalent inhibitory mechanism of nafamostat.

Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the precise functions and mechanisms of NM are not clear. In this study, we determined a series of crystal structures of NM and its hydrolysates in complex with a serine protease (urokinase-type plasminogen activator [uPA]). These structures reveal that NM was cleaved by uPA and that a hydrolyzed product (4-guanidinobenzoic acid [GBA]) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6-amidino-2-naphthol [6A2N]) released from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, the 6A2N side of intact NM binds to the specific pocket of uPA. Molecular dynamics simulations and end-point binding free-energy calculations show that the conf1 of NM (6A2N as P1 group) in the uPA-S195A:NM complex may be more stable than conf2 of NM (GBA as P1 group). Moreover, in the structure of uPA:NM complex, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for the further development of serine protease inhibitors.

High-Throughput Docking and Molecular Dynamics Simulations towards the Identification of Potential Inhibitors against Human Coagulation Factor XIIa.

Human coagulation factor XIIa (FXIIa) is a trypsin-like serine protease that is involved in pathologic thrombosis. As a potential target for designing safe anticoagulants, FXIIa has received a great deal of interest in recent years. In the present study, we employed virtual high-throughput screening of 500,064 compounds within Enamine database to acquire the most potential inhibitors of FXIIa. Subsequently, 18 compounds with significant binding energy (from -65.195 to -15.726 kcal/mol) were selected, and their ADMET properties were predicted to select representative inhibitors. Three compounds (Z1225120358, Z432246974, and Z146790068) exhibited excellent binding affinity and druggability. MD simulation for FXIIa-ligand complexes was carried out to reveal the stability and inhibition mechanism of these three compounds. Through the inhibition of activated factor XIIa assay, we tested the activity of five compounds Z1225120358, Z432246974, Z45287215, Z30974175, and Z146790068, with pIC50 values of 9.3∗10-7, 3.0∗10-5, 7.8∗10-7, 8.7∗10-7, and 1.3∗10-6 M, respectively; the AMDET properties of Z45287215 and Z30974175 show not well but have better inhibition activity. We also found that compounds Z1225120358, Z45287215, Z30974175, and Z146790068 could be more inhibition of FXIIa than Z432246974. Collectively, compounds Z1225120358, Z45287215, Z30974175, and Z146790068 were anticipated to be promising drug candidates for inhibition of FXIIa.

Insight to the residue in P2 position prevents the peptide inhibitor from being hydrolyzed by serine proteases.

Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA [Formula: see text] and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined KD and Ki of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket.

Improved therapeutic efficacy of quercetin-loaded polymeric nanoparticles on triple-negative breast cancer by inhibiting uPA.

Triple negative breast cancer (TNBC) is one kind of breast cancer that demonstrates highly aggressive tumor biology. The high heterogeneity of TNBC makes its individual clinical treatment extremely blind and limited, which also introduces more challenges into the diagnosis and treatment of diseases. Urokinase-type plasminogen activator (uPA) is a high level marker for breast cancer, which mediates tumor growth and metastasis. Quercetin is a plant-derived flavonoid in many plants, which inhibits uPA and has low bioavailability and mediocre pharmaceutical efficacy. Thus, we herein developed polymeric nanoparticulate systems from PLGA-TPGS (Qu-NPs) for quercetin oral delivery and evaluated the anticancer effect of this formulation on TNBC in vitro and in vivo. Qu-NPs have a uniform spherical morphology with a mean diameter of 198.4 ± 7.8 nm and good drug loading capacity (8.1 ± 0.4%). Moreover, Qu-NPs exhibited significantly improved inhibition on the growth and metastasis in TNBC cells. Following oral gavage, a remarkable antitumor effect of Qu-NPs on 4T1-bearing mice was observed with a tumor inhibition ratio of 67.88% and fewer lung metastatic colonies. Furthermore, the inhibitory effect of quercetin on the migration of uPA knockdown MDA-MB231 cells was greatly attenuated. Together, Qu-NPs improved the significant antitumor and antimetastatic effects by inhibiting uPA, which provides a new strategy for the treatment of TNBC.

Solution Structure of SpoIVB Reveals Mechanism of PDZ Domain-Regulated Protease Activity.

Intramembrane proteases hydrolyze peptide bonds within the cell membrane as the decision-making step of various signaling pathways. Sporulation factor IV B protease (SpoIVB) and C-terminal processing proteases B (CtpB) play central roles in cellular differentiation via regulated intramembrane proteolysis (RIP) process which activates pro-σK processing at the σK checkpoint during spore formation. SpoIVB joins CtpB in belonging to the widespread family of PDZ-proteases, but much remains unclear about the molecular mechanisms and structure of SpoIVB. In this study, we expressed inactive SpoIVB (SpoIVBS378A) fused with maltose binding protein (MBP)-tag and obtained the solution structure of SpoIVBS378A from its small angle X-ray scattering (SAXS) data. The fusion protein is more soluble, stable, and yields higher expression compared to SpoIVB without the tag. MBP-tag not only facilitates modeling of the structure in the SAXS envelope but also evaluates reliability of the model. The solution structure of SpoIVBS378A fits closely with the experimental scattering data (χ2= 1.76). Comparing the conformations of PDZ-proteases indicates that SpoIVB adopts a PDZ-protease pattern similar to the high temperature requirement A proteases (HtrAs) rather than CtpB. We not only propose that SpoIVB uses a more direct and simple way to cleave the substrates than that of CtpB, but also that they work together as signal amplifiers to activate downstream proteins in the RIP pathway.

Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris.

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human ßFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent.

Plasminogen activator inhibitor-1 (PAI-1) is a labile molecule that exists in four different forms: active, latent, cleaved and target bound form. Although there have been many methods to measure the total PAI-1, the measurement of active form of PAI-1 antigen is still challenging. Here we developed a novel ELISA to detect the active form of PAI-1 based on a highly specific PAI-1 capturing agent which binds to active PAI-1 with high affinity. We also used a highly stable PAI-1 mutant as an assay calibrator to enhance the method's reproducibility. This ELISA has the advantage of measuring both the antigen level and activity of PAI-1 at the same time. The assay had a sensitivity of 0.167 ng/ml and a working range of 0.195-25 ng/ml. The intra- and inter-assay variations were 6.7% and 11.3% respectively. The mean recovery of spiked standard was 102%. We used this strategy to measure the active PAI-1 level in plasma of healthy donors, and had an interesting observation: the PAI-1 level reduced by half after plasma storage for 6 h at room temperature. This finding represents the first observation of activity loss in plasma PAI-1 samples, and may explain large variations in PAI-1 levels (0-100 ng/ml) observed in human samples using commercial assays.

tPA Point Mutation at Autolysis Loop Enhances Resistance to PAI-1 Inhibition and Catalytic Activity.

Recombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.

A novel purification procedure for recombinant human serum albumin expressed in Pichia pastoris.

Plasma-derived human serum albumin (pHSA) has important applications in many clinical indications, including blood loss, serious burn, or hemorrhagic shock. The limited supply and potential infectious pathogen contamination of pHSA have stimulated the development of recombinant human serum albumin (rHSA). However, rHSA often entraps endogenous or exogenous impurities, including color pigments and polysaccharides. Therefore, the purification of rHSA to high purity remains the bottleneck in large-scale production of rHSA. Herein we report a novel two-step purification protocol for rHSA generated from Pichia pastoris. In the first step, rHSA was partially unfolded to expose impurities entrapped into rHSA and was then removed. In the second step, rHSA was crystallized to further remove impurities. Through this procedure, the pigment content (A350/A280) and polysaccharides content of rHSA were reduced to 0.0141 and 0.253 µg/mg, respectively, which were comparable to pHSA. In addition, the secondary structure and bioactivity of purified rHSA are preserved. The purification procedure developed in this study is a simple, short and low cost method to produce active rHSA, or rHSA-fusion proteins, to high purity, and also suitable for the purification of other disulfide-rich proteins.

Molecular basis of rutin inhibition of protein disulfide isomerase (PDI) by combined in silico and experimental methods.

Protein disulfide isomerase (PDI) is a founding member of the thiol isomerase family, and is recently found to play critical roles in thrombus formation. The development of effective PDI inhibitors is of great significance, and attracts strong interest. We previously showed that rutin bound directly to PDI and inhibited PDI activities, leading to the suppression of platelet aggregation and fibrin generation in a mouse model. A close analog of rutin, isoquercetin, is currently in advanced phase clinical trials. However, the molecular interaction between rutin and PDI is unknown and is difficult to study by X-ray crystallography due to the weak interaction. Here, we generated a molecular model of PDI:rutin complex by molecular docking and thorough molecular dynamics (MD) simulations. We then validated the complex model through a number of different experimental methods. We mutated the key residues predicted by the model and analyzed the mutants by an optimized isothermal titration calorimetry (ITC) method and a functional assay (insulin reduction assay). The results consistently showed that the PDI residues H354, L355 and E359 are important in the binding of rutin. These residues are next to the canonical major substrate binding site of the b' domain, and were not conserved across the members of thiol isomerases, explaining the specificity of rutin for PDI among vascular thiol isomerases. Furthermore, the inhibitory activities of three rutin analogues were evaluated using an insulin reduction assay. The results supported that the second sugar ring at the side chain of rutin was not necessary for the binding to PDI. Together, this work provides the structural basis for the inhibitory mechanism of rutin to PDI, and offers a promising strategy for the design of new generation inhibitors with higher binding affinity to PDI for therapeutic applications.

A structural mechanism of flavonoids in inhibiting serine proteases.

Quercetin is a member of the flavonoids and was previously demonstrated to inhibit trypsin-like serine proteases at micromolar potencies. Different molecular models were proposed to explain such inhibition. However, controversies remain on the molecular details of inhibition. Here, we report the X-ray crystal structure of quercetin in a complex with the urokinase-type plasminogen activator (uPA), an archetypical serine protease. The structure showed that quercetin binds to the specific substrate binding pocket (S1 pocket) of uPA mainly through its two neighboring phenolic hydroxyl groups. Our study thus provides unambiguous evidence to support quercetin binding to serine proteases and defines the molecular basis of the interaction. Our results further establish that natural products with two adjacent phenolic hydroxyl groups (or catechol) are likely to inhibit other trypsin-like serine proteases, a new mechanism formerly under-recognized.

A long-acting PAI-1 inhibitor reduces thrombus formation.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.

A specific plasminogen activator inhibitor-1 antagonist derived from inactivated urokinase.

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re-establish blood flow after thrombus formation. Plasminogen activator inhibitor-1 (PAI-1) inhibits urokinase-type and tissue-type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI-1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI-1 and identified a potent PAI-1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active-site mutation (S195A) and four additional mutations (G37bR-R217L-C122A-N145Q). PAItrap inhibits human recombinant PAI-1 with high potency (Kd = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI-1. In addition, PAItrap inhibits both human and murine PAI-1, allowing the evaluation in murine models. In vivo, using a laser-induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI-1 inhibitors using inactivated urokinase.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

[Indication and clinical radical effect of functional neck dissection for head and neck neoplasm].

BACKGROUND & OBJECTIVE: The functional neck dissection was first described by Bocca in 1967. It has been used on clinical practice for years. But still controversial between supporters and opponents of functional neck dissection. This study was designed to evaluate application and radical effectiveness of functional neck dissection(FND). METHODS: One hundred and fifty-two cases underwent FND were reviewed with analysis of pathological specimen and follow-up data. RESULT: One hundred and fifty-two cases of FND include tongue carcinoma 20 cases, larynx carcinoma 23 cases, thyroid papillary adenocarcinoma 96 cases, follicular adenocarcinoma 9 cases, medullary adenocarcinoma 4 cases; Five years recurrence rate of tongue and larynx carcinoma was 12.5%, 14.3%, 40.0% and 20%, 16.7%, 50.0% in N0, N1, N2, respectively. 5 years recurrence rate of thyroid papillary adenocarcinoma is 6.5%, 7.1% in N1a, N1b, respectively. CONCLUSION: Functional neck dissection is suitable for head and neck neoplasm, was result in satisfactory radical effects.