Partial Purification of a Specific Plasminogen Activator from Porcine Parotid Glands

1974 ◽  
Vol 31 (03) ◽  
pp. 403-414 ◽  
Author(s):  
Terence Cartwright
Keyword(s):  
Nucleic Acid ◽  
Salivary Glands ◽  
Plasminogen Activator ◽  
Protein Interactions ◽  
Partial Purification ◽  
Protein Protein Interactions ◽  
Parotid Glands ◽  
Two Stage ◽  
Preliminary Characterization ◽  
Specific Inhibitors

SummaryA method is described for the extraction with buffers of near physiological pH of a plasminogen activator from porcine salivary glands. Substantial purification of the activator was achieved although this was to some extent complicated by concomitant extraction of nucleic acid from the glands. Preliminary characterization experiments using specific inhibitors suggested that the activator functioned by a similar mechanism to that proposed for urokinase, but with some important kinetic differences in two-stage assay systems. The lack of reactivity of the pig gland enzyme in these systems might be related to the tendency to protein-protein interactions observed with this material.

scholarly journals Hidden partners: Using cross-docking calculations to predict binding sites for proteins with multiple interactions

2018 ◽  
Author(s):  
Nathalie Lagarde ◽  
Alessandra Carbone ◽  
Sophie Sacquin-Mora
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Protein Protein Interactions ◽  
Protein Binding Sites ◽  
Cross Docking ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Docking Calculations

AbstractProtein-protein interactions control a large range of biological processes and their identification is essential to understand the underlying biological mechanisms. To complement experimental approaches, in silico methods are available to investigate protein-protein interactions. Cross-docking methods, in particular, can be used to predict protein binding sites. However, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. We evaluate the binding site predictions obtained using complete cross-docking simulations of 358 proteins with two different scoring schemes accounting for multiple binding sites. Despite overall good binding site prediction performances, 68 cases were still associated with very low prediction quality, presenting individual area under the specificity-sensitivity ROC curve (AUC) values below the random AUC threshold of 0.5, since cross-docking calculations can lead to the identification of alternate protein binding sites (that are different from the reference experimental sites). For the large majority of these proteins, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, i.e. partners not included in the original cross-docking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, for proteins with multiple binding sites on their surface, we investigated the structural determinants associated with the binding sites the most targeted by the docking partners.AbbreviationsANOVA: ANalysis Of Variance; AUC: Area Under the Curve; Best Interface: BI; CAPRI: Critical Assessment of Prediction of Interactions; CC-D: Complete Cross-Docking; DNA: DesoxyriboNucleic Acid; FDR: False Discovery Rate; FRIres(type): Fraction of each Residue type in the Interface; FP: False Positives; GI: Global Interface; HCMD: Help Cure Muscular Dystrophy; JET: Joint Evolutionary Tree; MAXDo: Molecular Association via Cross Docking; NAI: Nucleic Acid Interface; NPV: Negative Predicted Value; PDB: Protein Data Bank; PIP: Protein Interface Propensity; PiQSi: Protein Quaternary Structure investigation; PPIs: Protein-Protein Interactions; PPV: Positive Predicted Value; Prec.: Precision; PrimI: Primary Interface; RNA: RiboNucleic Acid; ROC: Receiver Operating Characteristic; SecI: Secondary Interface; Sen.: Sensitivity; Spe.: Specificity; TN: True Negatives; TP: True Positives; WCG: World Community Grid.

scholarly journals Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

2016 ◽  
Vol 90 (9) ◽  
pp. 4593-4603 ◽  
Author(s):  
Tibor Füzik ◽  
Růžena Píchalová ◽  
Florian K. M. Schur ◽  
Karolína Strohalmová ◽  
Ivana Křížová ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Protein Interactions ◽  
Viral Genome ◽  
Virus Assembly ◽  
Protein Protein Interactions ◽  
Genome Packaging ◽  
Particle Assembly ◽  
Nuclear Trafficking ◽  
Virus Particles ◽  
Gag Polyprotein

ABSTRACTThe Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity usingin vitroandin vivosystems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle.IMPORTANCEAssembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

scholarly journals Generation of specific inhibitors of SUMO-1– and SUMO-2/3–mediated protein-protein interactions using Affimer (Adhiron) technology

2017 ◽  
Vol 10 (505) ◽  
pp. eaaj2005 ◽  
Author(s):  
David J. Hughes ◽  
Christian Tiede ◽  
Natalie Penswick ◽  
Anna Ah-San Tang ◽  
Chi H. Trinh ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Specific Inhibitors

scholarly journals Nucleic Acid Binding-Induced Gag Dimerization in the Assembly of Rous Sarcoma Virus Particles In Vitro

2004 ◽  
Vol 78 (1) ◽  
pp. 52-60 ◽  
Author(s):  
Yu May Ma ◽  
Volker M. Vogt
Keyword(s):  
Nucleic Acid ◽  
Protein Interactions ◽  
Rous Sarcoma Virus ◽  
Minimum Length ◽  
Protein Protein Interactions ◽  
Ph Change ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag (ΔMBDΔPR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of ΔMBDΔPR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of ΔMBDΔPR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.

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