The Alternatively Spliced Acid Box Region Plays a Key Role in FGF Receptor Autoinhibition

SummaryUncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition

Haplotype Analysis of RAGE Gene Polymorphisms and Association with Increased Risk of Diabetic Nephropathy

Background: The present study aimed at evaluating the association between the -429T/C and - 374T/A polymorphisms of RAGE (Receptor for Advanced Glycation End Products) gene promoter and diabetic nephropathy as well as examining its possible application as candidate markers of diabetic nephropathy among the population of Qazvin, Iran. Methods: In this study, the diabetic patients were divided into the two groups of with or without nephropathy. The frequency of genotype and allele were determined using TETRA-Primer ARMSPCR. Hardy-Weinberg equilibrium test and correlation of polymorphisms, odds ratio (OR), and FAMHAP software were used for haplotype analysis. Results: Based on our data, the CC genotype of -429T/C polymorphism may play a protective role against the development of nephropathy (OR=0.586, 95%; CI: 0.158-2.167) while, the AA genotype may be associated with increased risk of the disease (OR=1.889, 95%; CI: 0.454-7.854). Allele’s analysis revealed that the C allele of -429T/C polymorphism maybe protective against the appearance of nephropathy (OR=0.794, 95%; CI: 0.48-1.314) whereas, the A allele may be related to increased risk for nephropathy (OR=1.452, 95%; CI: 0.783-2.695). Haplotype analysis demonstrated that there was no significant correlation between the two -429T/C and -374T/A SNPs (χ2=5.125, p value=0.135). However, it was found that the CA haplotype may have a protective effect against the development of nephropathy (OR=0.48, 95%; CI: 0.14-1.64) while, the TA haplotype may increase the risk of the disease (OR=2.06, 95%; CI:1.01-4.23). Conclusion: Overall, no correlation between the -374T/A and -429T/C polymorphisms and the haplotypes in RAGE gene and the occurrence of diabetic nephropathy, was established. Keywords: Nephropathy, Type 2 Diabetes, Haplotype, Receptor for Advanced Glycation End Products, SNP, Iran Citation: Tavakoli A, Salahshourifar I, Hajialilo

A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1

Molecular Features and Properties of Mycobacterial Proteins Linked to Tuberculosis Pathogenesis

The Mycobacterium tuberculosis genome codes for 11 pairs of CFP-10/ESAT-6 proteins (Esx family) as well as the apparatus required for secretion of these proteins. The core machinery for the secretion of ESAT-6 and CFP-10 is encoded by their surrounding genes. Recent studies also identified a distant region, the Rv3612c-Rv3616c operon, which is essential for the CFP-10/ESAT-6 secretion. Constructs carrying Rv3613c, Rv3614c, Rv3615c and Rv3616c coding regions were produced and used to express the corresponding proteins. However, only Rv3614c and Rv3615c were expressed using an E. coli-based expression system. Analysis using a range of spectroscopic techniques on the purified proteins revealed that both Rv3615c and Rv3614c contain stable secondary structure, but little if any stable tertiary structure and exist in a molten globule-like state. This suggests the proteins probably undergo folding upon binding with possible functional partners. Yeast-two hybrid studies showed no intermolecular interaction between the proteins encoded by the Rv3616c-Rv3612c operon, perhaps suggesting the formation of a higher order multi-protein complex. Together with CFP-10 and ESAT-6, Rv0287 and Rv0288 are the members of the Esx family which are clearly implicated in M. tuberculosis pathogenesis. The expression vectors carrying Rv0287 and Rv0288 coding regions were constructed and used to express the proteins. Analysis using a range of spectroscopic techniques on the purified proteins showed that Rv0288 contains up to 30 % helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, Rv0287 has been found to form an unstructured, random coil polypeptide. The work reported here also shows that Rv0287 and Rv0288 form a tight 1:1 complex which is predominantly helical. Furthermore, the Rv0287-Rv0288 complex was found to be significantly more stable to thermal denaturation than CFP-10-ESAT-6. The high resolution solution structure reported here reveals that both proteins, Rv0287 and Rv0288, adopt an elongated helix-turn-helix hairpin structure in which the proteins lie antiparallel to each other, forming a stable four helix bundle. Comparison of the CFP-10-ESAT-6 and Rv0287-Rv0288 complexes also revealed that the overall backbone fold for the complexes is very similar although they display significantly different surface features

Molecular features and properties of mycobacterial proteins linked to tuberculosis pathogenesis

The Mycobacterium tuberculosis genome codes for 11 pairs of CFP-10/ESAT-6 proteins (Esx family) as well as the apparatus required for secretion of these proteins. The core machinery for the secretion of ESAT-6 and CFP-10 is encoded by their surrounding genes. Recent studies also identified a distant region, the Rv3612c-Rv3616c operon, which is essential for the CFP-10/ESAT-6 secretion. Constructs carrying Rv3613c, Rv3614c, Rv3615c and Rv3616c coding regions were produced and used to express the corresponding proteins. However, only Rv3614c and Rv3615c were expressed using an E. coli-based expression system. Analysis using a range of spectroscopic techniques on the purified proteins revealed that both Rv3615c and Rv3614c contain stable secondary structure, but little if any stable tertiary structure and exist in a molten globule-like state. This suggests the proteins probably undergo folding upon binding with possible functional partners. Yeast-two hybrid studies showed no intermolecular interaction between the proteins encoded by the Rv3616c-Rv3612c operon, perhaps suggesting the formation of a higher order multi-protein complex. Together with CFP-10 and ESAT-6, Rv0287 and Rv0288 are the members of the Esx family which are clearly implicated in M. tuberculosis pathogenesis. The expression vectors carrying Rv0287 and Rv0288 coding regions were constructed and used to express the proteins. Analysis using a range of spectroscopic techniques on the purified proteins showed that Rv0288 contains up to 30 % helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, Rv0287 has been found to form an unstructured, random coil polypeptide. The work reported here also shows that Rv0287 and Rv0288 form a tight 1:1 complex which is predominantly helical. Furthermore, the Rv0287-Rv0288 complex was found to be significantly more stable to thermal denaturation than CFP-10-ESAT-6. The high resolution solution structure reported here reveals that both proteins, Rv0287 and Rv0288, adopt an elongated helix-turn-helix hairpin structure in which the proteins lie antiparallel to each other, forming a stable four helix bundle. Comparison of the CFP-10-ESAT-6 and Rv0287-Rv0288 complexes also revealed that the overall backbone fold for the complexes is very similar although they display significantly different surface features.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase

Objective(s): Involvement of tyrosinase in the synthesis of melanin and cell signaling pathway has made it an attractive target in the search for therapeutic inhibitors for treatment of different skin hyperpigmentation disorders and melanoma cancers. Materials and Methods: In the present study, we conducted a comprehensive kinetic analysis to understand the mechanisms of inhibition imposed by 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, and picolinic acid on the monophenolase and diphenolase activities of the mushroom tyrosinase, and then MTT assay was exploited to evaluate their toxicity on the melanoma cells. Results: Kinetic analysis revealed that nicotinic acid and picolinic acid competitively restricted the monophenolase activity with inhibition constants (Ki) of 1.21 mM and 1.97 mM and the diphenolase activity with Kis of 2.4 mM and 2.93 mM, respectively. 2-aminobenzoic acid and 4-aminobenzoic acid inhibited the monophenolase activity in a non-competitive fashion with Kis of 5.15 μM and 3.8 μM and the diphenolase activity with Kis of 4.72 μM and 20 μM, respectively. Conclusion: Our cell-based data revealed that only the pyridine derivatives imposed cytotoxicity in melanoma cells. Importantly, the concentrations of the inhibitors leading to 50% decrease in the cell density (IC50) were comparable to those causing 50% drop in the enzyme activity, implying that the observed cytotoxicity is highly likely due to the tyrosinase inhibition. Moreover, our cell-based data exhibited that the pyridine derivatives acted as anti-proliferative agents, perhaps inducing cytotoxicity in the melanoma cells through inhibition of the tyrosinase activities