Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.     

The single NqrB and NqrC subunits in the Na(+)-translocating NADH: Quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Proteasome protease mediated regulation of cytokine induction and inflammation

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin-proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three‐dimensional structures with ～1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen‐sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (～1 µm). Two fluorophores, oxygen‐sensitive Ru(Ph₂phen₃)Cl₂, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 2663–2670. © 2012 Wiley Periodicals, Inc.     

Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics

α-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type (WT) protein in vitro and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure compared with wild-type AS. Here, we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length WT fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to determine its secondary structure empirically. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved β-sheet core.     

Conformational Changes in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase upon Substrate Binding: ROLE OF N-TERMINAL LOBE AND ENANTIOMERIC SUBSTRATE PREFERENCE

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP(5). Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP(5) 2-K, which shed light on aspects of substrate recognition. However, failure of IP(5) 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP(5) 2-K in its different conformations by crystallography. Thus, the IP(5) 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP(5) 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg(130) mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP(5) 2-K in mammals.     

The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.     

Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.