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Suppressor Of Cytokine Signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

The Alpha-1-Antitrypsin (A1AT) protein is an important protease inhibitor highly abundant in human serum and other body fluids. Additional to functioning as a protease inhibitor, A1AT is an important acute phase protein. Here, we set out to compare the proteoform profiles of A1AT purified from the human serum and milk of eight healthy donors to determine the origin of human milk A1AT. Following affinity purification, size-exclusion chromatography coupled to native mass spectrometry was used to monitor individual proteoform profiles comparing inter- and intra-donor profiles. The A1AT intra-donor proteoform profiles were found to be highly identical between serum and milk, while they were highly distinct between donors, even when comparing only serum or milk samples. The observed inter-donor proteoform variability was due to differences in the abundances of different N-glycoforms, mainly due to branching, fucosylation, and the relative abundance of N-terminally processed A1AT fragments. From our data we conclude that nearly all A1AT in serum and milk is synthesized by a common source, i.e. the liver, and then secreted into the circulation and enters the mammary gland via diffusion or transport. Thereby, proteoform profile changes, as seen upon infection and/or inflammation in the blood will be reflected in the milk, which may then be transferred to the breastfed infant.

Context Long-term weight loss (WL) maintenance is the biggest challenge for overweight and obesity because of the almost unavoidable phenomenon of partial or even total weight regain (WR) after WL. Objective In the present study we investigated the relations of (the changes of) adipocyte size and other risk biomarkers with WR during the follow-up of the Yoyo dietary intervention. Methods In this randomized controlled study, 48 overweight/obese participants underwent a very-low-calorie diet to lose weight, followed by a weight-stable period of 4 weeks and a follow-up period of 9 months. Anthropometric measurements, adipocyte volume of abdominal subcutaneous adipose tissue, and plasma metabolic parameters (free fatty acids [FFAs], triglycerides [TGs], total cholesterol, glucose, insulin, homeostasis model assessment of insulin resistance [HOMA-IR], interleukin 6 [IL-6], angiotensin-converting enzyme [ACE] activity, retinol binding protein 4 [RBP4]) at the beginning and the end of follow-up were analyzed. Results Our results show that changes of TGs, IL-6, HOMA-IR, and ACE are significantly positively correlated with WR. Multiple linear regression analysis shows that only TG and IL-6 changes remained significantly correlated with WR and increased body fat mass. Moreover, the change in HOMA-IR was tightly correlated with the change in TGs. Surprisingly, change in adipocyte volume during follow-up was not correlated with WR nor with other factors, but positive correlations between adipocyte volume and HOMA-IR were found at the beginning and end of the follow-up. Conclusion These results suggest that TGs and IL-6 are independently linked to WR via separate mechanisms, and that HOMA-IR and adipocyte volume may indirectly link to WR through the change of plasma TGs.

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of Significance: The challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.

More than 50 cytokines signal via the JAK/STAT pathway to orchestrate hematopoiesis, induce inflammation and control the immune response. Cytokines are secreted glycoproteins that act as intercellular messengers, inducing proliferation, differentiation, growth, or apoptosis of their target cells. They act by binding to specific receptors on the surface of target cells and switching on a phosphotyrosine-based intracellular signaling cascade initiated by kinases then propagated and effected by SH2 domain-containing transcription factors. As cytokine signaling is proliferative and often inflammatory, it is tightly regulated in terms of both amplitude and duration. Here we review molecular details of the cytokine-induced signaling cascade and describe the architectures of the proteins involved, including the receptors, kinases, and transcription factors that initiate and propagate signaling and the regulatory proteins that control it.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5′-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases., AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.

Given the association of disturbances in non-esterified fatty acid (NEFA) metabolism with the development of Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease, computational models of glucose-insulin dynamics have been extended to account for the interplay with NEFA. In this study, we use arteriovenous measurement across the subcutaneous adipose tissue during a mixed meal challenge test to evaluate the performance and underlying assumptions of three existing models of adipose tissue metabolism and construct a new, refined model of adipose tissue metabolism. Our model introduces new terms, explicitly accounting for the conversion of glucose to glyceraldehye-3-phosphate, the postprandial influx of glycerol into the adipose tissue, and several physiologically relevant delays in insulin signalling in order to better describe the measured adipose tissues fluxes. We then applied our refined model to human adipose tissue flux data collected before and after a diet intervention as part of the Yoyo study, to quantify the effects of caloric restriction on postprandial adipose tissue metabolism. Significant increases were observed in the model parameters describing the rate of uptake and release of both glycerol and NEFA. Additionally, decreases in the model’s delay in insulin signalling parameters indicates there is an improvement in adipose tissue insulin sensitivity following caloric restriction., Author summary The adipose tissue is no longer considered a metabolically quiescent tissue. Disturbances in non-esterified fatty acid (NEFA) metabolism leading to ectopic fat deposition have been associated with the development of insulin resistance. In recent years, the use of stable isotope tracers coupled with arteriovenous sampling across tissue depots has greatly improved our knowledge of postprandial NEFA dynamics. In this study, we make use of arteriovenous measurements collected across the abdominal subcutaneous adipose tissue in humans during a high fat mixed meal to evaluate three existing computational models of adipose tissue metabolism. As the three models included in this study were not capable of fully describing the measured adipose tissue fluxes, we present a new model of human in vivo adipose tissue metabolism, introducing novel terms such as the postprandial uptake of glycerol. We then utilised our refined model to quantify the effect of caloric restriction on adipose tissue metabolism by fitting the model to mixed meal challenge test data collected before and after a weight-loss intervention. Parameter estimates indicate an increase in the rates of glycerol and NEFA release coupled with a decrease in the delay of insulin signalling for all reactions, suggesting improved insulin sensitivity following caloric restriction.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4β1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications.

Crystal structures of human and prokaryotic ribosomal oxygenases reported here, with and without their ribosomal protein substrates, support their assignments as hydroxylases, and provide insights into the evolution of the JmjC-domain-containing hydroxylases and demethylases. Christopher Schofield and colleagues present a comprehensive structural study of a recently discovered family of 2-oxoglutarate and iron-dependent oxygenases termed ribosomal oxygenases (ROXs). They have solved 13 new structures of human and bacterial ROXs with or without substrate. While the analysis confirms their assignment as hydroxylases, comparison with JmjC-domain-containing hydroxylases and demethylases provides an understanding of the evolution of these related families of proteins, in part driven by flexibility in the coordination position. The general binding mode of the hydroxylated residues is conserved between prokaryotic and human ROXs. 2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Ne-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

SOCS3 (Suppressor of Cytokine Signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus Kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced poly-ubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalysed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

JAKs (Janus kinases) are essential mediators of almost all biological signalling events initiated by haemopoietic and immune cytokines. However, aberrant and/or prolonged JAK-induced signalling is detrimental and can give rise to a number of inflammatory and proliferative pathologies. For this reason, the tyrosine kinase activity of the JAKs is carefully regulated at a number of different levels. Primarily, this is achieved by: (i) ensuring that the catalytic domain is ‘switched off’ under basal conditions, and (ii) inhibiting the activity of JAK after it has been switched on. Whereas the first mode of inhibition is mediated by JAK's own pseudokinase domain as well as the action of phosphatases, the second is achieved by the action of the SOCS (suppressor of cytokine signalling) proteins, negative-feedback inhibitors of JAK-mediated signalling. The present review focuses on the mode of action of SOCS1 and SOCS3, the two most potent JAK inhibitors. Abbreviations: G-CSF, granulocyte colony-stimulating factor; gp130, glycoprotein 130; Grb14, growth factor receptor-bound protein 14; IL, interleukin; IRK, insulin receptor kinase; JAK, Janus kinase; JH1, JAK homology 1; KIR, kinase-inhibitory region; LIF, leukaemia-inhibitory factor; OSM, oncostatin M; SH2, Src homology 2; SOCS, suppressor of cytokine signalling; STAT, signal transducer and activator of transcription; TYK2, tyrosine kinase 2

Motivation Microscopy imaging is an essential tool for medical diagnosis and molecular biology. It is particularly useful for extracting information about disease states, tissue heterogeneity and cell specific parameters such as cell type or cell size from biological specimens. However, the information obtained from the images is likely to be subjected to sampling and observational bias with respect to the underlying cell size/type distributions. Results We present an algorithm, Estimate Tissue Cell Size/Type Distribution (EstiTiCS), for the adjustment of the underestimation of the number of small cells and the size of measured cells while accounting for the section thickness independent of the tissue type. We introduce the sources of bias under different tissue distributions and their effect on the measured values with simulation experiments. Furthermore, we demonstrate our method on histological sections of paraffin-embedded adipose tissue sample images from 57 people from a dietary intervention study. This data consists of measured cell size and its distribution over the dietary intervention period at four time points. Adjusting for the bias with EstiTiCS results in a closer fit to the true/expected adipocyte size distribution with earlier studies. Therefore, we conclude that our method is suitable as the final step in estimating the tissue wide cell type/size distribution from microscopy imaging pipeline. Availability and Implementation Source code and its documentation are available at https://github.com/michaelLenz/EstiTiCS. The whole pipeline of our method is implemented in R and makes use of the ‘nloptr’ package. Adipose tissue data used for this study are available on request. Contact Michael.Lenz@Maastrichtuniversity.nl, Gokhan.Ertaylan@Maastrichtuniversity.nl

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1-6, D1-D6) at 3.6 A resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4-D6) at 1.9 A. This represents the first atomic resolution structure of the complete ectodomain of any "tall" cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.

The 40-residue peptide isoform beta-amyloid (Abeta(1-40)) is associated with Alzheimer's disease. Although found in the tangles and fibrous mats that characterize the brain in advanced stages of the disease, the toxic form of Abeta is believed to be oligomers or "protofibrils". Characterization of these fairly small structures in solution, especially in the presence of the much larger assemblies they also form, is a daunting task. Additionally, little is known about the rate of Abeta assembly or whether it can be triggered easily. Perhaps most importantly, the conditions for reversing assembly are not fully understood. Fluorescence photobleaching with modulation detection of the recovery profile is a sensitive and materials-efficient way to measure diffusers over a wide range of hydrodynamic sizes. The method does require attachment of a fluorescent label. Experiments to validate the use of 5-carboxyfluorescein-labeled Abeta(1-40) as a representative of the unlabeled, naturally occurring material included variation of photobleaching time and mixture of labeled and unlabeled materials. A dialysis cell facilitated rapid in situ changes in pH and salt conditions. Multiple steps and complex protocols can be explored with relative ease. Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes.

(3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A β-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P21 space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley-Liss, Inc.

The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ≈ 15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to > 95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate α and β subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an α2β2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the β subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the β subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major β subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate, but not the formation of N-acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N-acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the β subunit.

Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.

SummaryAging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases.

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/beta-lactamase-type fold with highest structural similarity to the class A beta-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show beta-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, beta-lactamases and d-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

The changes in phospholipid compositions, membrane fluidity and protein kinase A, protein kinase C, tyrosine and casein kinase activities in chick embryo liver plasma membranes during development have been investigated. The percentage participation of sphingomyelin increased while that of phosphatidylserine decreased during chick embryo development. The alterations in membrane sphingomyelin accompanied an increase of steady-state fluorescence anisotropy (rs) of membrane bilayer. Regression analysis indicated positive linear correlations between the percentage participation of sphingomyelin in total membrane phospholipids and (i) protein kinase C (r = 0.903); (ii) casein kinase (r = 0.936); (iii) protein kinase A (r = 0.850); (iv) tyrosine kinase (r = 0.960) activities. We suggest that sphingomyelin might be an specific activator for all types of protein kinase activities investigation.

A comprehensive analysis of the phosphoinositide interactome has been performed using an ω-amino analogue of phosphatidylinositol 3-phosphate (PI(3)P immobilised onto Affi-10 beads for use as an affinity absorbent for cytosolic, membrane and nuclear extracts from the LIM1215 colonic carcinoma cell line. Affinity/LC/MS/MS experiments allowed the identification of 681 proteins/protein complexes which interact with PI(3)P. Protein domain enrichment analysis identified proteins possessing PI(3)P (e.g., FYVE, PX, PH), PIP and PIP/phospholipid binding domains along with small GTPases, GTPase regulators, kinases and SH2/SH3 containing proteins. Functional and pathway enrichment analyses highlighted the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, intracellular cell signalling regulation, cell division and cytokinesis. Biological significance This study provides an initial detailed assessment of the phosphatidylinositol 3-phosphate (PI(3)P) interactome, highlights the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, cell intracellular regulation, signalling and cytokinesis and suggests potential PI(3)P specificity for further biochemical and biological characterisation.

The inhibitory protein SOCS3 plays a key part in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between mouse SOCS3, JAK2 (kinase domain) and a fragment of the interleukin-6 receptor β-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. While the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phosphoindependent manner to a noncanonical surface. The kinase-inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2, and biochemical studies show that it blocks substrate association. These studies reveal that SOCS3 targets specific JAK-cytokine receptor pairs and explains the mechanism and specificity of SOCS action.

Cytosol cAMP-independent quercetin-inhibited protein kinase of developing chick embryo liver was measured at three embryonic ages (days 12, 14 and 18) in the presence of casein and poly (Glu-Na, Tyr) 4:1 as substrates. In the early embryonic stages the tyrosine kinase was almost as active as casein kinase, but on day 18 the tyrosine phosphorylation was only 25% of the casein phosphorylation. Both kinase activities strongly increased by the end of embryonic development: 7-fold with casein and 2.6-fold with poly (Glu-Na, Tyr) 4:1. Triiodothyronine caused twice the stimulation of casein and tyrosine phosphorylation on day 12, but had no effect on day 18.

β-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although β-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of β-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular β-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of β-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of β-catenin within this complex. Each component of β-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential β-catenin-interacting proteins, and define when and where a specific complex forms.

Background The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration. Findings In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions. Conclusions We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.

1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter (rs) of the fluorescence probe DPH and SDPH decreased strongly in the experimental group. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.

The influence of the phospholipid composition and fluidity on protein kinase A and protein kinase C activities in rat liver plasma membrane was studied. We observed that enrichment of membranes with phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and dioleoylphosphatidylcholine caused activation of both protein kinases. Phosphatidylglycerol was found to be most effective activator. The enrichment of plasma membranes with dipalmitoylphosphatidylcholine and sphingomyelin led to decrease in protein kinase A and C activities. The stimulatory effect of phosphatidylglycerol was confirmed in plasma membranes pretreated with exogenous phospholipases A 2 , C and D, and subsequently enriched with phosphatidylglycerol. We suggest that besides the specific presence of definite phospholipids protein kinases A and C require a more fluid membrane lipid bilayer to display an optimal activity.

Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.

The ultimate step in the biosynthesis of the medicinally important beta-lactamase inhibitor clavulanic acid is catalyzed by clavulanic acid dehydrogenase (CAD). CAD is responsible for the NAPDH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to yield clavulanic acid. Here, we report biochemical and structural studies on CAD. Biophysical analyses demonstrate that CAD exists as dimeric and tetrameric species in solution. The reaction performed by CAD was shown to be reversible, allowing the use of clavulanic acid for activity analyses. The crystal structure of CAD was solved using single-wavelength anomalous diffraction with a seleno-methionine derivative. The structure reveals that the individual monomers comprise a single domain possessing the Rossmann fold, characteristic of dinucleotide-binding enzymes. The monomers are arranged as tetramers, similar to other tetrameric members of the short-chain dehydrogenase/reductase family. The structure of the unreactive complex of CAD with clavulanic acid and NADPH suggests how CAD is able to catalyze the reduction of clavulanate-9-aldehyde without fragmentation of the bicyclic beta-lactam ring structure. The relative positions of NADPH and clavulanic acid, in the active site, together with the presence of the latter in an eclipsed conformation, rationalizes previous labeling studies demonstrating that the incorporation of the C5 pro-R, but not pro-S, hydrogen of ornithine/arginine into the C9 position of clavulanic acid occurs with overall inversion of configuration.

The beta-amyloid peptide aggregates via a nucleation pathway where micellar aggregates propagate to form oligomers (protofibrils), which then polymerize into insoluble fibrils. This fibrillogenic process has been linked to the pathogenesis associated with Alzheimer's disease. One purpose of this chapter is to provide a protocol for reliably producing monomeric Abeta as a starting point for physical and biological studies. Many research groups have used organic solvents to disaggregate pre-seeded Abeta in an attempt to acquire monomeric starting materials. Others have used instrumental techniques such as size exclusion chromatography to isolate monomer, structural intermediates, and fibrils and study their affects on Abeta nucleation. This chapter discusses a modified method of Abeta preparation using organic solvents followed by dissolution into aqueous phosphate buffer systems that renders monomeric Abeta starting solutions for kinetic experiments. Additionally, this chapter details a number of physical techniques such as scanning force microscopy, circular dichroism spectroscopy, transmission electron microscopy, fluorescence spectroscopy, fluorescence photobleaching recovery, and dynamic light scattering, together with physiological techniques such as cell viability assays to characterize Abeta nucleation, aggregation, and fibrillization and the potential biological activity of the various Abeta particles.

The enzyme-catalysed reactions involved in formation of the bicyclic clavam and carbapenem nuclei, including beta-amino acid and beta-lactam formation, are discussed and compared with those involved in penicillin and cephalosporin biosynthesis. The common role of unusual oxidation reactions in the biosynthetic pathways and the lack of synthetic reagents available to effect them are highlighted.

Mononuclear non-heme ferrous iron dependent oxygenases and oxidases constitute an extended enzyme family that catalyze a wide range of oxidation reactions. The largest known sub-group employs 2-oxoglutarate as a cosubstrate and catalysis by these and closely related enzymes is proposed to proceed via a ferryl intermediate coordinated to the active site via a conserved HXD/E … H motif. Crystallographic studies on the 2-oxoglutarate oxygenases and related enzymes have revealed a common double-stranded β-helix core fold that supports the residues coordinating the iron. This fold is common to proteins of the cupin and the JmjC transcription factor families. The crystallographic studies on 2-oxoglutarate oxygenases and closely related enzymes are reviewed and compared with other metallo-enzymes/related proteins containing a double-stranded β-helix fold. Proposals regarding the suitability of the active sites and folds of the 2-oxoglutarate oxygenases to catalyze reactions involving reactive oxidizing species are described.

(3R,5R)-Clavulanic acid, a clinically used inhibitor of serine beta-lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic beta-lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis.

Phytanic acid is a 3-methyl branched fatty acid that cannot be degraded by P-oxidation. Instead, one carbon atom is removed to give pristanic acid by a single round of peroxisomal a-oxidation. Phytanoyl-CoA 2- hydroxylase (PAHX), an iron(II) and 2-oxoglutarate oxygenase, catalyses the first step in this pathway. Defects in PAHX cause some forms of adult Refsum’s disease, in which phytanic acid accumulates (Wanders et al., 2001; Wierzbicki et al., 2002).

SPRY/B30.2 domain-containing proteins are found in 103 human proteins that regulate numerous cellular processes including RNA processing, histone methylation, red blood cell membrane integrity, terminal erythroid cell differentiation and innate immune responses. Despite an increased understanding of SPRY/B30.2 domain function, some confusion remains as to the precise domain boundaries, the number and location of binding sites, and importantly how activation of these domains contribute to signal transduction. The B30.2 domain of tripartite motif 25 (TRIM25) interacts with the viral RNA sensor retinoic acid-inducible gene I (RIG-I), facilitating the formation of a RIG-I tetramer, which is required for activation of the innate immune response against RNA viruses such as influenza A, measles, HIV and hepatitis C. Here we have investigated the biochemical and structural changes associated with the activation of TRIM25 in the context of RIG-I binding. We solved a 1.8 Å crystal structure of the TRIM25 B30.2 domain and identified a putative RIG-I-binding site, which was confirmed by mutagenesis and functional analyses. Further mutagenesis identified a second site on the opposing face of the B30.2 domain that also specifically interacts with RIG-I. While the CARD1 domain of RIG-I is known to interact with the B30.2 domain of TRIM25, our data suggest that a conformational change within the CARD2 domain of RIG-I enables it to also interact with the B30.2 domain of TRIM25. We propose that both RIG-I CARDs interact with sites on opposing sides of the TRIM25 B30.2 domain to form a TRIM25/RIG-I complex that facilitates RIG-I tetramerisation and activation. The characterization of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family. Disease-associated polymorphisms of Pyrin, involved in Familial Mediterranean Fever, and mutational analyses of TRIM5a, required for HIV restriction, suggest that a second binding site may be a characteristic feature of other B30.2-containing TRIM proteins, and may facilitate the formation of large oligomeric complexes that are important for inflammasome function and HIV capsid destruction. Disclosures No relevant conflicts of interest to declare.