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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.

Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied., Young et al. report on the diversity in regulation of nematode apoptosis. Results indicate that the apoptotic pathways of C. elegans are not fully representative of nematodes generally.

Learning rules by which cell shape impacts cell function would enable control of cell physiology and fate in medical applications, particularly, on the interface of cells and material of the implants. We defined the phenotypic response of human bone marrow-derived mesenchymal stem cells (hMSCs) to 2176 randomly generated surface topographies by probing basic functions such as migration, proliferation, protein synthesis, apoptosis, and differentiation using quantitative image analysis. Clustering the surfaces into 28 archetypical cell shapes, we found a very strict correlation between cell shape and physiological response and selected seven cell shapes to describe the molecular mechanism leading to phenotypic diversity. Transcriptomics analysis revealed a tight link between cell shape, molecular signatures, and phenotype. For instance, proliferation is strongly reduced in cells with limited spreading, resulting in down-regulation of genes involved in the G2/M cycle and subsequent quiescence, whereas cells with large filopodia are related to activation of early response genes and inhibition of the osteogenic process. In this paper we were aiming to identify a universal set of genes that regulate the material-induced phenotypical response of human mesenchymal stem cells. This will allow designing implants that can actively regulate cellular, molecular signalling through cell shape. Here we are proposing an approach to tackle this question.

The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines., Cytokines are key molecules in controlling haematopoiesis that signal via the JAK/STAT pathway. Here the authors present the structures of SOCS1 bound to its JAK1 target as well as in complex with elonginB and elonginC, providing a molecular explanation for the potent JAK- inhibitory activity of SOCS1.

Objective During weight loss, the volume of adipocytes decreases, leading to stress because of the misfit between the cell contents and the surrounding extracellular matrix (ECM). This stress can be resolved by remodeling the ECM or the restorage of triglycerides within the adipocytes. The objective of this study was to investigate the existence of a connection between stress-related and ECM-related genes that is associated with weight regain. Methods Thirty-one participants with overweight or obesity followed a 5-week very-low-calorie diet (500 kcal/d) with a subsequent 4-week weight-stable diet (WS), and then an uncontrolled 9-month follow-up. Adipose tissue biopsies were collected for microarray analysis. A correlation and interaction analysis was performed with the weight regain percentage (WR%) ([weight after follow-up - weight after WS] ÷ weight after WS × 100%) by using two gene sets that were previously defined as "stress-related" (n = 107) and "ECM-related" genes (n = 277). Results During WS, a coexpression network of 8 stress-related genes and 15 ECM-related genes correlating with WR% could be constructed, with links to multiple biological processes. Interaction analysis between stress- and ECM-related genes revealed that several gene combinations were highly related to weight regain. Conclusions Our findings underscore the importance of the connection between stress- and ECM-related genes in the risk for weight regain.

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.

Background: Weight loss (WL) is often followed by weight regain after an energy-restricted dietary intervention (DI). When people are following a diet, the volume of an adipocyte decreases by loss of triglycerides, which creates stress between the cell contents and the surrounding extracellular matrix (ECM). Previously, we observed that genetic variations in ECM genes are associated with an increased risk of weight regain. Objective: We investigated the relation between the expression of ECM genes during WL and a period of weight stabilization (WS) and the risk of weight regain. Design: In this randomized controlled trial, 61 healthy overweight or obese participants followed either a 5-wk very-low-calorie diet (VLCD; 500 kcal/d) or a 12-wk low-calorie diet (1250 kcal/d) (WL period) with a subsequent 4-wk WS period and a 9-mo follow-up. The WL and WS periods combined were considered the DI. Abdominal subcutaneous adipose tissue biopsy samples were collected for microarray analysis. Gene expression changes for a broad set of ECM-related genes were correlated with the weight-regain percentage (WR%). Results: A total of 26 of the 277 genes were significantly correlated with WR% during WL, WS, or the DI periods. Most correlations were observed in the VLCD group during the WS period. Four genes code for leukocyte-specific receptors. These and other genes belong to a group of 26 genes, among which the expression changes were highly correlated (r ≥ 0.7, P ≤ 0.001). This group could be divided into 3 subclusters linking to 2 biological processes-leukocyte integrin gene activity and ECM remodeling-and a link to insulin sensitivity was also apparent. Conclusions: Our present findings indicate the importance of adipose tissue leukocytes for the risk of weight regain. ECM modification also seems to be involved, and we observed a link to insulin sensitivity. This trial was registered at clinicaltrials.gov as NCT01559415.

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.

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.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer’s disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Primary cilia are organelles that are present on many different cell types, either transiently or permanently. They play a crucial role in receiving signals from the environment and passing these signals to other parts of the cell. In that way, they are involved in diverse processes such as adipocyte differentiation and olfactory sensation. Mutations in genes coding for ciliary proteins often have pleiotropic effects and lead to clinical conditions, ciliopathies, with multiple symptoms. In this study, we reviewed observations from ciliopathies with obesity as one of the symptoms. It shows that variation in cilia-related genes is itself not a major cause of obesity in the population but may be a part of the multifactorial aetiology of this complex condition. Both common polymorphisms and rare deleterious variants may contribute to the obesity risk. Genotype–phenotype relationships have been noticed. Among the ciliary genes, obesity differs with regard to severity and age of onset, which may relate to the influence of each gene on the balance between pro- and anti-adipogenic processes. Analysis of the function and location of the proteins encoded by these ciliary genes suggests that obesity is more linked to activities at the basal area of the cilium, including initiation of the intraflagellar transport, but less to the intraflagellar transport itself. Regarding the role of cilia, three possible mechanistic processes underlying obesity are described: adipogenesis, neuronal food intake regulation and food odour perception.

Initial successful weight loss is often followed by weight regain after the dietary intervention. Compared with lean people, cellular stress in adipose tissue is increased in obese subjects. However, the relation between cellular stress and the risk for weight regain after weight loss is unclear. Therefore, we determined the expression levels of stress proteins during weight loss and weight maintenance in relation to weight regain. In vivo findings were compared with results from in vitro cultured human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes. In total, eighteen healthy subjects underwent an 8-week diet programme with a 10-month follow-up. Participants were categorised as weight maintainers or weight regainers (WR) depending on their weight changes during the intervention. Abdominal subcutaneous adipose tissue biopsies were obtained before and after the diet and after the follow-up. In vitro differentiated SGBS adipocytes were starved for 96 h with low (0·55 mm) glucose. Levels of stress proteins were determined by Western blotting. WR showed increased expressions of β-actin, calnexin, heat shock protein (HSP) 27, HSP60 and HSP70. Changes of β-actin, HSP27 and HSP70 are linked to HSP60, a proposed key factor in weight regain after weight loss. SGBS adipocytes showed increased levels of β-actin and HSP60 after 96 h of glucose restriction. The increased level of cellular stress proteins in the adipose tissue of WR probably resides in the adipocytes as shown by in vitro experiments. Cellular stress accumulated in adipose tissue during weight loss may be a risk factor for weight regain.

Tau inclusions are a shared feature of many neurodegenerative conditions and tau mutations lead to frontotemporal dementia. Approaches to treatment of these conditions have focused directly on the tau protein by targeting its post-translational modifications, its levels and its tendency to aggregate. We discovered a novel regulatory pathway for tau degradation that operates through the Rhes protein, a GTPase. Rhes is farnesylated and treatment with the farnesyl transferase inhibitor, lonafarnib, reduced Rhes, attenuated behavioral abnormalities, significantly reduced atrophy, tau inclusions, sumoylation and ubiquitination, as well as microgliosis in the rTg4510 tauopathy mouse. Direct reduction of Rhes levels reproduced the results observed with lonafarnib. The mechanism of lonafarnib action, as mediated by Rhes to reduce tau pathology, operates through the lysosome without involvement of the proteasome. Finally we show that the developmental increase in Rhes levels can be homeostatically regulated in the presence of tau mutations as a protective mechanism through which cells sense abnormal tau before any pathology is present. The extensive human trials of lonafarnib for other conditions, makes this drug ideal for repurposing to treat tauopathies.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Stress during pregnancy increases the risk to develop psychological disorders such as depression during pregnancy or in the postpartum period. According to the neurotrophin hypothesis of depression, the pathophysiology of depression is caused by reduced neurotrophic activity in the brain. However, most studies only focus on the molecular changes happening to the offspring upon gestational stress. To gain insight into the potential molecular changes happening in the stressed dams, C57B16/J mice were stressed during their first week of gestation. At 28 days postpartum, the hippocampus and nucleus accumbens core of the dams, two brain regions heavily implicated in depression, were evaluated using immunohistochemistry to detect changes in the neurotrophin system. Gestational stress decreased the weight of the dams, increased the chance for spontaneous abortion and increased the weight of offspring. Litter size, survival rates and sex distribution were not altered as a consequence of gestational stress. Hippocampal brain-derived neurotrophic factor (BDNF) decreased following exposure to stress during pregnancy. Hippocampal protein levels of p75(NTR), a low-affinity receptor for BDNF which can induce apoptosis, were increased following exposure to stress. Protein levels of p11, of which the expression is regulated by BDNF, were decreased in the hippocampus. No changes were found for TrkB immunostaining or apoptosis. Taken together, this shows that stress during pregnancy negatively affects the neurotrophin system in the hippocampus of the dams, thereby reducing hippocampal plasticity. These data confirm that gestational stress has a negative impact on pregnancy. Open Access funded by VSNU

Adipose tissue autophagy (AT) is associated with human obesity and increased metabolic risk. Recent findings establish a role for autophagy in lipid metabolism. Here, we compared the expression of autophagy-related and lipolysis genes in human abdominal subcutaneous AT (SCAT) in overweight/obese subjects (n = 17) with or without impaired glucose tolerance in comparison with lean normal glucose tolerant individuals (n = 9), and investigated the association between AT autophagy and lipolysis. Human multipotent adipose-derived stem cells (hMADS) were used to investigate the effect of pharmacological HSL inhibition on changes in the autophagic flux. The expression of autophagy-related genes (ATG) 5, 7 and 12 in SCAT was significantly higher (p = 0.043, p = 0.015, p = 0.004, respectively) in overweight/obese compared to lean men, while expression of the classical lipases HSL (p = 0.092) and ATGL (p = 0.084) tended to be lower. ATG12 mRNA was positively correlated with BMI (r = 0.407, p = 0.039). ATG7 mRNA correlated positively with waist/hip ratio (WHR) (r = 0.420, p = 0.041), 2 h glucose concentration (r = 0.488, p = 0.011) and insulin (r = 0.419, p = 0.033). Multiple linear regressions revealed that ATG7 gene expression was positively related to 2 h glucose, independent of BMI, WHR and insulin. Gene expression interaction analysis showed that ATG7 mRNA negatively correlated with HSL (p

Three Alternaria radicina (Meier) Neerg isolates (A1, A2, and A3) were isolated and firstly recorded in date palm, Phoenix dactylifera L. leaves in Wasit province (middle of Iraq) and identified as the causative pathogen of black spot disease. Two fungicides (Bavistin and Tachigazole) were used in vitro to measure their ability to control this pathogen. Study results revealed that both chemical fungicides expressed high level of inhibition in fungal radial growth especially at a concentration of 100 ppm and the lowest growth was at the concentration 5 ppm among all three isolates in both two fungicides. Bavistin revealed higher inhibition rate than Tachigazole with lowest LC50 (12.21) at the isolate A3. Tachigazole treatment showed high level of variation at all concentrations.

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.

Janus kinases (JAKs) initiate the intracellular signaling cascade triggered by exposure of cells to cytokines and interferons. In order to achieve this, JAKs are bound to the intracellular domain of specific cytokine receptors immediately adjacent to the cell membrane. In this issue of Structure, Ferrao et al. (2016) provide structural details of such an interaction and in doing so, identify for the first time the motif used by type II cytokine receptors to recruit JAK1.

It is so frustrating to finally lose some weight only to gain it back almost immediately. Research conducted at Maastricht University provides new insight into the way this yo-yo effect works. When we lose weight, our fat cells lose some of the fat they have stored. As a result, the cytoskeleton does not match the cell volume anymore. That is why the fat cells will develop stress. A fat cell can lose stress by hormonally stimulating the host to eat more and store fat in the fat cells. This will lead to weight gain. In addition to stress in fat cells, the immune system plays a role in this process, too.

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.

SummaryJanus kinases (JAKs) are key effectors in controlling immune responses and maintaining hematopoiesis. SOCS3 (suppressor of cytokine signaling-3) is a major regulator of JAK signaling and here we investigate the molecular basis of its mechanism of action. We found that SOCS3 bound and directly inhibited the catalytic domains of JAK1, JAK2, and TYK2 but not JAK3 via an evolutionarily conserved motif unique to JAKs. Mutation of this motif led to the formation of an active kinase that could not be inhibited by SOCS3. Surprisingly, we found that SOCS3 simultaneously bound JAK and the cytokine receptor to which it is attached, revealing how specificity is generated in SOCS action and explaining why SOCS3 inhibits only a subset of cytokines. Importantly, SOCS3 inhibited JAKs via a noncompetitive mechanism, making it a template for the development of specific and effective inhibitors to treat JAK-based immune and proliferative diseases.

Obesity is a global epidemic identified as a major risk factor for multiple chronic diseases and, consequently, diet-induced weight loss is used to counter obesity. The adipose tissue is the primary tissue affected in diet-induced weight loss, yet the underlying molecular mechanisms and changes are not completely deciphered. In this study, we present a network biology analysis workflow which enables the profiling of the cellular processes affected by weight loss in the subcutaneous adipose tissue. Time series gene expression data from a dietary intervention dataset with two diets was analysed. Differentially expressed genes were used to generate co-expression networks using a method that capitalises on the repeat measurements in the data and finds correlations between gene expression changes over time. Using the network analysis tool Cytoscape, an overlap network of conserved components in the co-expression networks was constructed, clustered on topology to find densely correlated genes, and analysed using Gene Ontology enrichment analysis. We found five clusters involved in key metabolic processes, but also adipose tissue development and tissue remodelling processes were enriched. In conclusion, we present a flexible network biology workflow for finding important processes and relevant genes associated with weight loss, using a time series co-expression network approach that is robust towards the high inter-individual variation in humans.

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.

BACKGROUND: Energy restriction causes adaptations in energy expenditure (total-,TEE; resting-,REE; activity induced-,AEE). OBJECTIVE: To determine if changes in the levels of proteins involved in adipocyte glucose and fatty acid metabolism as indicators for energy deficiency are related to adaptations in energy expenditure during weight loss. METHODS: Forty-eight healthy subjects (18 men, 30 women), mean +/- SD age 42 +/- 8 y and BMI 31.4 +/- 2.8 kg/m(2), followed a very low energy diet for 8 wk. Protein levels of fatty acid binding protein 4 (FABP4), fructose-bisphosphate aldolase C (AldoC) and short chain 3-hydroxyacyl-CoA dehydrogenase (HADHsc) (adipose tissue biopsy, western blot), TEE (doubly labeled water), REE (ventilated hood), and AEE were assessed before and after the 8-wk diet. RESULTS: There was a positive correlation between the decrease in AldoC and the decrease in TEE (R = 0.438, P < 0.01) and the decrease change in AEE (R = 0.439, P < 0.01). Furthermore, there was a negative correlation between the increases in HADHsc and the decrease in REE (R = 0.343, P < 0.05). CONCLUSION: The decrease in AldoC correlated with the decrease in AEE, which may be explained by a decreased glycolytic flux. Additionally, the change in HADHsc, a crucial enzyme for a step in beta-oxidation, correlated with the adaptation in REE. TRIAL REGISTRATION: CLINICAL TRIAL REGISTRATION NUMBER: NCT01015508 at clinicaltrials.gov.

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses. DOI: http://dx.doi.org/10.7554/eLife.08698.001, eLife digest The immune system protects the body from invading microbes like bacteria and viruses. Upon recognizing the presence of these microbes, cells in the immune system are activated to destroy the foreign threat and clear it from the body. A type of immune cell called T follicular helper cells (or Tfh for short) are formed during an infection and are essential for coordinating other immune cells to produce high-quality antibody proteins that attack the microbes. Without Tfh cells, life-long production of these protective antibodies is severely crippled, which can cause common variable immune deficiency and other serious immunodeficiency diseases. On the other hand, the body must also avoid generating excessive numbers of Tfh cells, which can lead to the production of antibodies that attack healthy cells of the body. ROQUIN is a protein that inhibits the formation of Tfh cells and other types of active T cells. A region on the protein called the ROQ domain destabilizes particular molecules of ribonucleic acid (RNA) that are required for these specialist T cells to form and work properly. ROQUIN belongs to a large family of enzymes that have a so-called RING domain, which is a feature that enables these enzymes to attach tags onto specific target proteins to modify their activity or stability. However, it was not known whether the RING domain of ROQUIN was active. Ramiscal et al. now address this question in mice. Unexpectedly, the experiments show that the RING domain is required to promote the formation of Tfh cells, but not other types of active T cells. This domain allows ROQUIN to repress an enzyme called AMPK, which normally blocks cell growth by regulating cell metabolism. The findings suggest that the different roles of the ROQ and RING domains allow ROQUIN to fine-tune the numbers of Tfh cells so that they remain within a safe range. In the future, these findings may aid the development of vaccines that are more efficient at generating protective Tfh cells to prevent infectious diseases. DOI: http://dx.doi.org/10.7554/eLife.08698.002

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.

BACKGROUND/OBJECTIVES: Moderate weight loss (WL) can ameliorate adverse health effects associated with obesity, reflected by an improved adipose tissue (AT) gene expression profile. However, the effect of rate of WL on the AT transcriptome is unknown. We investigated the global AT gene expression profile before and after two different rates of WL that resulted in similar total WL, and after a subsequent weight stabilization period.SUBJECTS/METHODS: In this randomized controlled trial, 25 male and 28 female individuals (body mass index (BMI): 28-35 kg m(-)(2)) followed either a low-calorie diet (LCD; 1250 kcal day(-1)) for 12 weeks or a very-low-calorie diet (VLCD; 500 kcal day(-1)) for 5 weeks (WL period) and a subsequent weight stable (WS) period of 4 weeks. The WL period and WS period together is termed dietary intervention (DI) period. Abdominal subcutaneous AT biopsies were collected for microarray analysis and gene expression changes were calculated for all three periods in the LCD group, VLCD group and between diets (ΔVLCD-ΔLCD).RESULTS: WL was similar between groups during the WL period (LCD: -8.1±0.5 kg, VLCD: -8.9±0.4 kg, difference P=0.25). Overall, more genes were significantly regulated and changes in gene expression appeared more pronounced in the VLCD group compared with the LCD group. Gene sets related to mitochondrial function, adipogenesis and immunity/inflammation were more strongly upregulated on a VLCD compared with a LCD during the DI period (positive ΔVLCD-ΔLCD). Neuronal and olfactory-related gene sets were decreased during the WL period and DI period in the VLCD group.CONCLUSIONS: The rate of WL (LCD vs VLCD), with similar total WL, strongly regulates AT gene expression. Increased mitochondrial function, angiogenesis and adipogenesis on a VLCD compared with a LCD reflect potential beneficial diet-induced changes in AT, whereas differential neuronal and olfactory regulation suggest functions of these genes beyond the current paradigm.International Journal of Obesity advance online publication, 7 December 2016; doi:10.1038/ijo.2016.201.

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.

The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B (EloB), and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison to other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and the rationale design of novel compounds that may modulate cullin–substrate receptor interactions.

Valeriana officinalis (Valerianaceae) has been of great interest for its therapeutic uses for treating mild nervous tension and temporary sleeping problems. In traditional European medicine it has been also reported as an antiinflammatory remedy. This study reports that the EtOAc extract of the underground parts of V. officinalis showed inhibitory activity against NF-κB at 100 µg/mL in the IL-6/Luc assay on HeLa cells and provided protection against excitotoxicity in primary brain cell cultures at micromolar concentrations. Bioassay-guided fractionation of the EtOAc extract led to the isolation of three known sesquiterpenes: acetylvalerenolic acid (1), valerenal (2) and valerenic acid (3), 1 and 3 were active as inhibitors of NF-κB at a concentration of 100 µg/mL. Acetylvalerenolic acid (1) reduced NF-κB activity to 4%, whereas valerenic acid (3) reduced NF-κB activity to 25%. Copyright © 2006 John Wiley & Sons, Ltd.

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 eight SOCS (Suppressor of Cytokine Signaling) proteins encoded in the human genome all contain a C-terminal domain, the SOCS box, that allows them to function as E3 ubiquitin ligases and thereby catalyze the ubiquitination of components of the JAK/STAT signaling pathway. This activity is key to their function as cytokine signaling inhibitors as, once ubiquitinated, signaling molecules are degraded by the proteasome which allows the cell to return to its basal (unstimulated) state. SOCS based E3s are a subset of the CRL (Cullin-Ring-Ligase) family of ubiquitin ligases with the SOCS protein acting as the substrate recruitment module and interacting specifically with Cullin5, the E3 scaffold. Included here are protocols for the expression and purification of SOCS-based E3 complexes and their use in in vitro ubiquitination assays to characterize potential substrates. We have currently verified two components of the JAK/STAT pathway as substrates for ubiquitination using this method.

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.

Bivalve molluscs are newly discovered models of successful aging. Here, we test the hypothesis that extremely long-lived bivalves are not uniquely resistant to oxidative stressors (eg, tert-butyl hydroperoxide, as demonstrated in previous studies) but exhibit a multistress resistance phenotype. We contrasted resistance (in terms of organismal mortality) to genotoxic stresses (including topoisomerase inhibitors, agents that cross-link DNA or impair genomic integrity through DNA alkylation or methylation) and to mitochondrial oxidative stressors in three bivalve mollusc species with dramatically differing life spans: Arctica islandica (ocean quahog), Mercenaria mercenaria (northern quahog), and the Atlantic bay scallop, Argopecten irradians irradians (maximum species life spans: >500, >100, and ~2 years, respectively). With all stressors, the short-lived A i irradians were significantly less resistant than the two longer lived species. Arctica islandica were consistently more resistant than M mercenaria to mortality induced by oxidative stressors as well as DNA methylating agent nitrogen mustard and the DNA alkylating agent methyl methanesulfonate. The same trend was not observed for genotoxic agents that act through cross-linking DNA. In contrast, M mercenaria tended to be more resistant to epirubicin and genotoxic stressors, which cause DNA damage by inhibiting topoisomerases. To our knowledge, this is the first study comparing resistance to genotoxic stressors in bivalve mollusc species with disparate longevities. In line with previous studies of comparative stress resistance and longevity, our data extends, at least in part, the evidence for the hypothesis that an association exists between longevity and a general resistance to multiplex stressors, not solely oxidative stress. This work also provides justification for further investigation into the interspecies differences in stress response signatures induced by a diverse array of stressors in short-lived and long-lived bivalves, including pharmacological agents that elicit endoplasmic reticulum stress and cellular stress caused by activation of innate immunity.

The aim of the present study was to develop a seasonality palynological model from modern feces of Lama guanicoe for the interpretation of pollen found in coprolites. For this purpose, fecal samples of L. guanicoe were collected during a year from dung piles located in the ecotone Subantarctic Forest-Patagonian Steppe, Perito Moreno National Park (PMNP) (NW Santa Cruz province, Argentina). For every season, the food items were characterized and the post-depositional pollen contamination of feces was evaluated. The results showed differences among the pollen sets of feces from every season and no post-depositional pollen contamination of the superficial fecal subsamples. Pollen concentration and percentage values evidenced high pollen abundance of: a) Asteraceae subfam. Asteroideae-Senecio type, Mulinum spinosum and Rumex in summer feces; b) Poaceae in autumn feces; c) Empetrum rubrum in winter feces; and d) Nassauvia, Plantago and Poaceae in spring feces. Seasonality variations in the feces were related to the phenology of plants, to variations in the diet and to the supply of palatable resources for L. guanicoe. This seasonal feces model will be useful as an analog in studies on Holocene camelid coprolites found in Cerro Casa de Piedra (PMNP) sites. It will allow clarifying the seasonality in site use. On the other hand, it will contribute data to the L. guanicoe´s diet, which represent an important information for the species´ regional sustainable management. Fil: Velázquez, Nadia Jimena. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Palinología y Bioantropología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina Fil: Burry, Lidia Susana. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Palinología y Bioantropología; Argentina

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.