Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling

In comparison with pyeloplasty, endourologic procedures for the treatment of ureteropelvic junction obstruction offer good success rates with less morbidity and a shorter hospitalization; however, studies have found lower success rates and increased complications in patients with crossing vessels. Conventional diagnostic angiography and intravenous urography have both been used to identify crossing vessels at the UPJ; but, a reliable, less invasive, less costly, and simpler preoperative procedure to identify crossing vessels is needed. Helical CT with CT angiography is a promising noninvasive technique for the identification of crossing vessels at the ureteropelvic junction, which can be used for surgical planning of endourologic treatment of UPJ obstruction.     

Comparative modeling of the three CP modules of the {beta}-chain of C4BP and evaluation of potential sites of interaction with protein S

A computer model of the ß-chain of C4b-binding protein(C4BP) was constructed, using the backbone fold of the NMR structuresof the sixteenth CP module of factor H (H16) and of a pair ofmodules consisting of the fifteenth and sixteenth CPs of factorH (H15-16). The characteristic hydrophobic core responsiblefor dictating the three-dimensional structure of the CP familyis conserved in the amino acid sequence of C4BP ßl, ß2and ß3. The distribution of the electrostatic potentialshows that the model is mainly covered by a negative contour.Interestingly, a positive area is observed in the C-terminalregion of the first CP module, enclosing peptide 31-45, knownto be a binding site for protein S. This observation suggeststhat electrostatic interactions can be of importance for theinteraction of C4BP to protein S. A solvent-accessible hydrophobicpatch, located nearby and involving the peptide 31-45, was alsofound in the model, further confirming that this area is involvedin the interaction with protein S. The contribution of ß-chainresidues 31-45 to the affinity for protein S was studied furtherby means of synthetic mutant peptides. The results suggest thatboth electrostatic and hydrophobic interactions are importantfor the binding to protein S.     

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.     

Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function

Human protein S (PS) potentiates the anticoagulant activity of human but not bovine activated protein C (APC), whereas bovine PS is a cofactor to APC from both species. The structural requirements for the specificity of the APC cofactor function of human PS are located in its thrombin-sensitive region (TSR) and the first epidermal growth factor (EGF1)-like module. To elucidate which residues in these two modules determine the specificity of the APC cofactor activity, 41 human PS mutants were expressed. All mutants were cofactors to human APC and some also to bovine APC. Residues in TSR (positions 49 and 52) and EGF1 (residues 97 and 106) together determined the specificity of the APC cofactor function, whereas substitution of individual residues did not change specificity. Bovine PS, and mutants expressing cofactor activity to bovine APC, stimulated phospholipid binding of bovine APC. In contrast, human PS and mutants lacking cofactor activity to bovine APC failed to support binding of bovine APC to phospholipids. These data indicate that residues in TSR and EGF1 cause the specificity of the APC cofactor activity and support the concept that key residues in these two modules interact with APC on the phospholipid surface.     

In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.     

Development of Novel Thiazolopyrimidines as CDC25B Phosphatase Inhibitors

CDC25 inhibition by thiazolopyrimidines : CDC25 is an attractive target for cancer therapy, as it is overexpressed in numerous cancers and is often associated with tumor aggressiveness and poor prognosis. Based on an in silico/in vitro screen, we developed a series of thiazolopyrimidines to further improve inhibitory activity and cytotoxic properties.

     

Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling

Neglected fractures of the femoral neck, common in young adults in underdeveloped countries, may be complicated by nonunion or avascular necrosis (AVN). We treated 52 cases by open reduction, fixation by compression screw and a free fibular graft. The mean delay between injury and operation was 5.1 months. Of 40 fractures assessed at a mean of 58.8 months (24 to 153), 38 were found to be united and two, owing to surgical errors, were not. Seven of eight heads which were avascular before operation revascularised without collapse, while seven others developed AVN after the procedure. At the last follow-up considerable collapse was apparent in five femoral heads, and 11 hips had developed coxa vara. The fibular graft had fractured in four cases. The hip had been penetrated by the screw in six cases and by the graft in three. Hip function was excellent in seven patients, good in 21 and fair in seven. Five patients had poor results. Incorporation of the fibular graft was seen after four years: in many cases the graft had been almost completely resorbed. We recommend this procedure for the treatment of neglected fractures of the neck of the femur in young adults to reduce resorption of the neck, AVN and nonunion.