Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ.

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

The Remarkable Roles of the Receptor for Advanced Glycation End Products (RAGE) and Its Soluble Isoforms in COVID-19: The Importance of RAGE Pathway in the Lung Injuries.

The respiratory symptoms of acute respiratory distress syndrome (ARDS) in the coronavirus disease 2019 (COVID-19) patients is associated with accumulation of pre-inflammatory molecules such as advanced glycation end-products (AGES), calprotectin, high mobility group box family-1 (HMGB1), cytokines, angiotensin converting enzyme 2 (ACE2), and other molecules in the alveolar space of lungs and plasma. The receptor for advanced glycation end products (RAGEs), which is mediated by the mitogen-activated protein kinase (MAPK), plays a critical role in the severity of chronic inflammatory diseases such as diabetes mellitus (DM) and ARDS. The RAGE gene is most expressed in the alveolar epithelial cells (AECs) of the pulmonary system. Several clinical trials are now being conducted to determine the possible association between the levels of soluble isoforms of RAGE (sRAGE and esRAGE) and the severity of the disease in patients with ARDS and acute lung injury (ALI). In the current article, we reviewed the most recent studies on the RAGE/ligands axis and sRAGE/esRAGE levels in acute respiratory illness, with a focus on COVID-19-associated ARDS (CARDS) patients. According to the research conducted so far, sRAGE/esRAGE measurements in patients with CARDS can be used as a powerful chemical indicator among other biomarkers for assessment of early pulmonary involvement. Furthermore, inhibiting RAGE/MAPK and Angiotensin II receptor type 1 (ATR1) in CARDS patients can be a powerful strategy for diminishing cytokine storm and severe respiratory symptoms.

Isolated Lactobacillus fermentum Ab.RS22 from traditional dairy products inhibits HeLa cervical cancer cell proliferation and modulates apoptosis by the PTEN-Akt pathway.

Objectives: It is worthwhile to note that, some probiotics such as Lactobacilli and Bifidobacteria isolated from dairy products have significant therapeutic effects against cancer cells. Here, we evaluated anti-proliferation and the apoptotic effects of isolated Lactobacillus fermentum Ab.RS22 from traditional dairy products on the HeLa cervical cancer cells in vitro. Materials and Methods: The viability of treated HeLa cells with supernatant of Lactobacillus in 0.5, 0.75, 1, 1.5, and 2 ng/ml concentrations, and IC50 values were detected by tetrazolium bromide. The L. fermentum Ab.RS22-induced cell death by flow cytometry was confirmed through evaluation of the expression of caspase-3, P53, PTEN, and AKT genes by quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Results: Most cytotoxicity effects of Lactobacillus on HeLa cells were detected in 2 ng/ml at 24 hr (P<0.01); also, the IC50 value was measured as 1.5 ng/ml. The findings of the flow cytometry assay showed that L. fermentum Ab.RS22 in 1.5 ng/ml concentration at 24 hr increased the percentage of both apoptosis and necrosis cells. Lactobacillus-induced cell death was verified through results of Real-time PCR; where expression of caspase-3, P53, and PTEN genes was increased (P<0.01), and also expression of AKT gene (anti-apoptotic) was decreased (P<0.05). Conclusion: Our findings showed that L. fermentum Ab.RS22 could dose-dependently inhibit the proliferation of the HeLa cells. Its apoptotic effect was confirmed via modulating PTEN/p53/Akt gene expression and activation of the caspase-3 mediated apoptosis pathway. Therefore, L. fermentum Ab.RS22 can be considered a valuable anticancer candidate against cervical cancer progression in subsequent studies.

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

Structural characterization of recombinant human fibroblast growth factor receptor 2b kinase domain upon interaction with omega fatty acids.

The mutated recombinant kinase domain of human fibroblast growth factor receptor 2b (hFGFR2b) is overexpressed and purified, and its structural changes upon the interaction with three unsaturated fatty acids (UFAs), oleic, linoleic and α-linolenic are studied. This interaction is investigated to find out about the folding and unfolding effect of unsaturated fatty acids on the kinase domain structure of hFGFR2b. Recombinant pLEICS-01 vectors, containing the mutated coding region of hFGFR2b, are expressed in the standard Escherichia coli BL21 (DE3) host cells and purified by Ni2+-NTA affinity chromatography. While polyacrylamide gel electrophoresis characterizes the functionality of recombinant protein, its structural changes are studied in the presence and absence of various concentrations of oleic, α-linolenic and linoleic acids using circular dichroism (CD) and fluorescence spectroscopy. Far ultraviolet CD results show that unsaturated fatty acids do not change the secondary structure of the recombinant kinase domain of hFGFR2b. However, chemical denaturation analysis confirms that all three UFAs destabilize the tertiary structure of recombinant protein. A decrease in the fluorescence intensity without any significant red or blue shift (336±1nm) reflects a variation in the tertiary structure of protein. The direct interaction of the studied UFAs with hFGFR2b reduces the conformational stability of their kinase domains. The structural changes in hFGFR2b in the presence of UFAs may be necessary for hFGFR2b to adjust the signal transduction and regulate the key cellular processes.

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach.

BACKGROUND: Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK) , and alpha interferon (IFN-α) preparations. METHODS: A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry. RESULTS: The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA. CONCLUSION: Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers' minds on refining drugs, and provides consumers with safer biopharmaceuticals.

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

OBJECTIVES: 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.

The alternatively spliced acid box region plays a key role in FGF receptor autoinhibition.

Uncontrolled 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.

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 CCl(4)-induced injury in rats compared to rhKGF-1.

A comprehensive structure-function analysis shed a new light on molecular mechanism by which a novel smart copolymer, NY-3-1, assists protein refolding.

An in-depth understanding of molecular basis by which smart polymers assist protein refolding can lead us to develop a more effective polymer for protein refolding. In this report, to investigate structure-function relationship of pH-sensitive smart polymers, a series of poly(methylacrylic acid (MAc)-acrylic acid (AA))s with different MAc/AA ratios and molecular weights were synthesized and then their abilities in refolding of denatured lysozyme were compared by measuring the lytic activity of the refolded lysozyme. Based on our analysis, there were optimal MAc/AA ratio (44% MAc), M(w) (1700 Da), and copolymer concentration (0.1%, w/v) at which the highest yield of protein refolding was achieved. Fluorescence, circular dichroism, and RP-HPLC analysis reported in this study demonstrated that the presence of P(MAc-AA)s in the refolding buffer significantly improved the refolding yield of denatured lysozyme without affecting the overall structure of the enzyme. Importantly, our bioseparation analysis, together with the analysis of zeta potential and particle size of the copolymer in refolding buffers with different copolymer concentrations, suggested that the polymer provided a negatively charged surface for an electrostatic interaction with the denatured lysozyme molecules and thereby minimized the hydrophobic-prone aggregation of unfolded proteins during the process of refolding.

(15)N, (13)C and (1)H resonance assignments and secondary structure determination of the Mycobacterium tuberculosis Rv0287-Rv0288 protein complex.

Comprehensive sequence specific (1)H, (15)N, and (13)C resonance assignments are reported for the Mycobacterium tuberculosis Rv0287-Rv0288 protein complex. Analysis of the chemical shift data obtained indicates that each protein in the complex contains two relatively long helical regions joined by an irregular loop.

Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins.

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.