Fibroblast growth factor 21 (FGF21) is an endocrine FGF that plays a vital role in regulating essential metabolic pathways. FGF21 increases glucose uptake by cells, promotes fatty acid oxidation, reduces blood glucose levels, and alleviates metabolic diseases. However, detailed studies on its stability and biophysical characteristics have not been reported. Herein, we present the overexpression, biophysical characterization, and metabolic activity of a soluble recombinant FGF21 (rFGF21). The far-UV circular dichroism spectra of rFGF21 show a negative trough at 215 nm, indicating that the protein's backbone predominantly adopts a 13 sheet conformation. rFGF21 shows intrinsic tyrosine fluorescence at 305 nm. Thermal denaturation using differential scanning calorimetry reveals that rFGF21 is relatively thermally unstable, with a melting temperature of 46.8 degrees C (50.1 degrees C). The urea-induced unfolding of rFGF21 is rapid, with a chemical transition midpoint of 0.4 M. rFGF21 is readily cleaved by trypsin in limited trypsin digestion assays. Isothermal titration calorimetry experiments show that rFGF21 does not bind to heparin. Interestingly, rFGF21 demonstrates proliferative activity in NIH/3T3 fibroblasts and enhances mitochondrial oxidative phosphorylation and fatty acid oxidation in 3T3-L1 adipocytes. These findings provide a crucial framework for the engineering of novel structure-based variants of FGF21 with improved stability and biological activity to treat metabolic disorders. WHY IT MATTERS Metabolic diseases, such as obesity and diabetes, are serious global health concerns. Fibroblast growth factor 21 (FGF21) is a key metabolic regulator mediating glucose and fatty acid metabolism. Thus, FGF21 has significant potential as a therapeutic agent for the treatment of diabetes and obesity. However, limited research is available on its structure and stability. Herein, we characterize the structures and stability of a recombinant FGF21 using biophysical methods. The results of this study provide valuable insights for the development of potent FGF21-based therapeutic agents to combat dilapidating metabolic diseases. Additionally, this study paves a way for more in-depth structural studies of FGF21 in the future and facilitates a better understanding of FGF21's mode of action.

The family of fibroblast growth factors (FGFs) consists of 22 members with diverse biological functions in cells, from cellular development to metabolism. The family can be further categorized into three subgroups based on their three modes of action. FGF19, FGF21, and FGF23 are endocrine FGFs that act in a hormone-like/endocrine manner to regulate various metabolic activities. However, all three members of the endocrine family require both FGF receptors (FGFRs) and klotho co-receptors to elicit their functions. α-klotho and β-klotho act as scaffolds to bring endocrine FGFs closer to their receptors (FGFRs) to form active complexes. Numerous novel studies about metabolic FGFs' structures, mechanisms, and physiological insights have been published to further understand the complex molecular interactions and physiological activities of endocrine FGFs. Herein, we aim to review the structures, physiological functions, binding mechanisms to cognate receptors, and novel biomedical applications of endocrine FGFs in recent years.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

The family of fibroblast growth factor (FGF) is responsible for meditating various biological functions: wound healing, angiogenesis and glucose regulations. The FGF family is composed of endocrine, paracrine and intracrine FGFs. The paracrine FGFs, like FGF1, work locally through FGF receptors and heparin sulfate (HS) to regulate crucial activities like cell growth, differentiation, and tissue repairs. FGF1 has been investigated extensively and gave rise to a FGF1-variant termed SuperFGF1 (sFGF1).

The modified nucleosides 2 '-deoxy-7-cyano- and 2 '-deoxy-7-amido-7-deazaguanosine (dPreQ(0) and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction-modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD-dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ(0)), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ(0) into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ(0)-modified DNA to ADG-modified DNA.

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus, with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development.

Fibroblast growth factor (FGF) family is consisted of 22 proteins that regulate a wide variety of biological functions such as wound healing, tissue repairs, and angiogenesis. FGF family follows three main modes of action: paracrine, intracrine or endocrine. One member of the paracrine FGFs is FGF1, which works locally through interaction with heparin or heparin sulfate (HS). With its mitogenic and cell survival activities, FGF1 participates in many developmental processes, cell growth and differentiation, tissue repairs, and tumor growth and invasion. A representative of endocrine FGFs is FGF21, which is often used in long range physiological processes, but does not require HS. FGF21 engages in many functions across various organs like liver, pancreas, adipose tissues. One of FGF21's abilities is the regulation of glucose uptake in adipocytes through upregulating the transcription of glucose transporter-1. Thus, FGF21 is a potential treatment for metabolic diseases such as Type-2 Diabetes. However, FGF21 has been reported to possess an inherently unstable core with low thermal stability and receptor affinity. Herein, we designed a structure-based chimera protein by replacing the core of FGF21 with a thermally stable paracrine FGF1 (sFGF1). Using techniques such as fluorescence spectroscopy, and circular dichroism, we show that the chimera protein sFGF1-FGF21 adopts a β-trefoil core. Intrinsic fluorescence spectra of the chimera protein shows an emission maximum centered at 307 nm indicating that the emission of the conserved tryptophan is quenched similar to wildtype FGF1. Differential calorimetry data suggests that chimera sFGF1-FGF21 is significantly more stable than wild type FGF21. The findings of this study provide valuable clues for the rational design of FGF21-based therapeutic against hyperglycemia and obesity.

Fibroblast growth factors (FGFs) are cell-signaling proteins with diverse functions in cell development, repair, and metabolism. The human FGF family consists of 22 structurally related members, which can be classified into three separate groups based on their action of mechanisms, namely: intracrine, paracrine/autocrine, and endocrine FGF subfamilies. FGF19, FGF21, and FGF23 belong to the hormone-like/endocrine FGF subfamily. These endocrine FGFs are mainly associated with the regulation of cell metabolic activities such as homeostasis of lipids, glucose, energy, bile acids, and minerals (phosphate/active vitamin D). Endocrine FGFs function through a unique protein family called klotho. Two members of this family, α-klotho, or β-klotho, act as main cofactors which can scaffold to tether FGF19/21/23 to their receptor(s) (FGFRs) to form an active complex. There are ongoing studies pertaining to the structure and mechanism of these individual ternary complexes. These studies aim to provide potential insights into the physiological and pathophysiological roles and therapeutic strategies for metabolic diseases. Herein, we provide a comprehensive review of the history, structure–function relationship(s), downstream signaling, physiological roles, and future perspectives on endocrine FGFs.

The specificity and potency of venom components give them a unique advantage in developing various pharmaceutical drugs. Though venom is a cocktail of proteins, rarely are the synergy and association between various venom components studied. Understanding the relationship between various components of venom is critical in medical research. Using meta-analysis, we observed underlying patterns and associations in the appearance of the toxin families. For Crotalus, Dis has the most associations with the following toxins: PDE; BPP; CRL; CRiSP; LAAO; SVMP P-I and LAAO; SVMP P-III and LAAO. In Sistrurus venom, CTL and NGF have the most associations. These associations can predict the presence of proteins in novel venom and understand synergies between venom components for enhanced bioactivity. Using this approach, the need to revisit the classification of proteins as major components or minor components is highlighted. The revised classification of venom components is based on ubiquity, bioactivity, the number of associations, and synergies. The revised classification can be expected to trigger increased research on venom components, such as NGF, which have high biomedical significance. Using hierarchical clustering, we observed that the genera’s venom compositions were similar, based on functional characteristics rather than phylogenetic relationships.

Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G+), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ0). In bacteria, preQ0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ0 is inserted directly in tRNA by the archaeal TGT before conversion to G+. Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G+ and preQ0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G+‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by >140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA (SmDpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn2+‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

The specificity and potency of venom components gives them a unique advantage in development of various pharmaceutical drugs. Though venom is a cocktail of proteins rarely is the synergy and association between various venom components studied. Understanding the relationship between various components is critical in medical research. Using meta-analysis, we found underlying patterns and associations in the appearance of the toxin families. For Crotalus, Dis has the most associations with the following toxins: PDE; BPP; CRL; CRiSP; LAAO; SVMP P-I & LAAO; SVMP P-III and LAAO. In Sistrurus venom CTL and NGF had most associations. These associations can be used to predict presence of proteins in novel venom and to understand synergies between venom components for enhanced bioactivity. Using this approach, the need to revisit classification of proteins as major components or minor components is highlighted. The revised classification of venom components needs to be based on ubiquity, bioactivity, number of associations and synergies. The revised classification will help in increased research on venom components such as NGF which have high medical importance.

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t6A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteria.

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.Support or Funding InformationThis work was supported by the National Science Foundation and by the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t6A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteria.