Structure–function correlations in tyrosinases

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure–function correlations in tyrosinases along with comparison to other type-3 copper proteins.     

Do thyroid hormones function in insects?

Earlier work demonstrated that phenoxy-phenyl compounds such as fenoxycarb and thyroxine mimicked the effects of JH III in causing a reduction in volume of the follicle cells of Locusta migratoria. While these compounds were only moderately effective, a derivative of thyroxine, 3,3',5-triiodothyronine (T3) was as effective as JH III, and T3 has been shown to bind to the same membrane receptor and activate the same pathway as JH III. The current paper shows that other thyroxine derivatives vary in activity. 3,3', 5'-Triiodothyronine (reverse T3) is inactive. 3,5-Diiodothyronine (T2) is more active than JH III, while its relatives (iodines at 3', 5' or at 3,3') are inactive. When follicles are exposed in vitro to rhodamine conjugated T3, the fluorescent compound can be seen to enter the cells and accumulate there: this process is inhibited by cycloheximide or by a temperature of 0 degrees C. The accumulation is antagonised by JH III but not JH I (which does not bind to the JH III membrane receptor) and by an antiserum raised against the putative membrane receptor protein. The action of T3, but not T2, is inhibited by 6-n-propyl-2-thiouracil or by aurothioglucose, both known to inhibit deiodinases. The activity of T3, but not of T2, increases with time of exposure to the follicle cells. These facts suggest that T3 enters the cells by receptor mediated endocytosis and is converted to a more active compound. Immunoreactivity to T3, but not thyroxine, can be detected in the haemolymph of locusts, and the titre varies slightly with the gonotrophic cycle. The food shows immunoreactivity for both thyroxine and T3. These findings suggest that thyroid hormones are ingested by locusts and have the potential to be used as hormonal signals in the control of egg production.     

Structure–function relationships in HIV-1 Nef

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure–function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.     

An ambiguity in the notion “function”

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An ambiguity in the notion function     

Does dietary DHA improve neural function in children? Observations in phenylketonuria

Children with phenylketonuria (PKU) have a restricted protein intake and thus low dietary intakes of long-chain polyunsaturated fatty acids (LC-PUFA), which may cause subtle neurological deficits. We measured plasma phospholipid fatty acids and visual evoked potential (VEP) in 36 children with well-controlled PKU (6.3±0.6 years, 19 girls), before and after 3 months of supplementing fish oil capsules providing 15 mg docosahexaenoic acid (DHA)/kg daily. The motometric Rostock-Oseretzky Scale (ROS) was performed before and after supplementation in the 24 PKU children aged >4 years. VEP latencies and ROS were also assessed in omnivorous, age-matched controls without fish oil supply at baseline and after 3 months. Fish oil supply increased plasma phospholipid eicosapentaenoic acid (EPA) (0.40±0.03 vs 3.31±0.19%, p<0.001) and DHA (2.37±0.10 vs 7.05±0.24%, p<0.001), but decreased arachidonic acid (AA) (9.26±0.23 vs 6.76±0.16%, p<0.001). Plasma phenylalanine was unchanged. VEP latencies and ROS results significantly improved after fish oil in PKU children, but remained unchanged in controls. The improvements of VEP latencies, fine motor and coordination skills indicate that preformed n-3 LC-PUFA are needed for neural normalcy in PKU children. The optimal type and dose of supply still needs to be determined. Since PKU children are generally healthy and have normal energy and fatty acid metabolism, these data lead us to conclude that childhood populations in general require preformed n-3 LC-PUFA to achieve optimal neurological function.     

TGFβ signalling in the development of ovarian function

Ovarian development begins back in the embryo with the formation of primordial germ cells and their subsequent migration and colonisation of the genital ridges. Once the ovary has been defined structurally, the primordial germ cells transform into oocytes and become housed in structures called follicles (in this case, primordial follicles), a procedure that, in most mammals, occurs either shortly before or during the first few days after birth. The growth and differentiation of follicles from the primordial population is termed folliculogenesis. Primordial follicles give rise to primary follicles that transform into preantral follicles, then antral follicles (secondary follicles) and, finally (preovulatory) Graafian follicles (tertiary follicles) in a co-ordinated series of transitions regulated by hormones and local intraovarian factors. Members of the transforming growth factor-β (TGFβ) superfamily have been shown to play important roles in this developmental process starting with the specification of primordial germ cells by the bone morphogenetic proteins through to the recruitment of primordial follicles by anti-Mullerian hormone and, potentially, growth and differentiation factor-9 (GDF9) and, finally, their transformation into preantral and antral follicles in response to activin and TGF-β. Developmental and mutant mouse models have been used to show the importance of this family of growth factors in establishing the first wave of folliculogenesis.The author thanks the NHMRC of Australia for funding (Regkey 241000).     

Structure–function relationships in human d-amino acid oxidase

Since d-amino acids were identified in mammals, d-serine has been one of the most extensively studied “unnatural amino acids”. This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of d-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of d-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and d-amino acid oxidase (which catalyzes d-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human d-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of d-amino acid oxidase in brain d-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.     

TNF-α system and lung function impairment in obesity

A potential interaction between pulmonary function, abnormal adipose tissue activity, and systemic inflammation has been suggested. This study explores the relationship between circulating soluble TNF-α receptors (sTNF-R1 and sTNF-R2) and respiratory function parameters in obese subjects. Thirty-one non-diabetic morbidly obese women with a history of non-smoking and without prior cardiovascular or respiratory disease were prospectively recruited in the outpatient Obesity Unit of a referral center. Pulmonary function test included a forced spirometry, static pulmonary volume measurements, non-attended respiratory polygraphy, and arterial gas blood sampling. Circulating levels of sTNFR-R1, sTNF-R2, interleukine 6 and adiponectin were determined using ELISA. Statistical analysis included a multivariate regression analysis taking into account the potential confounders. sTNF-R1 positively correlated with BMI (r=0.571, p=0.001) and arterial carbon dioxide pressure (PaCO(2), r=0.381, p=0.038), but negatively with forced expiratory volume in 1s (FEV(1), r=-0.437, p=0.012), maximum midexpiratory flow (FEF(25-75), r=-0.370, p=0.040) and forced vital capacity (FVC, r=-0.483, p=0.005). However, no correlation between sTNF-R2 and BMI and either pulmonary function tests or arterial blood samples was observed. Multiple linear regression analysis showed that sTNF-R1 independently predicted FEV(1) (beta=-0.437, p=0.012) and FVC (beta=-0.483, p=0.005). Thus, circulating levels of sTNF-R1, but not sTNF-R2, are related to reduced lung volumes and airflow limitation in morbidly obese patients prior to the development of a clinically recognized respiratory disease. Therefore, studies addressed to evaluating the potential beneficial effect of anti-TNF-α agents on pulmonary function tests in obese subjects seem warranted.     

How do Rab proteins function in membrane traffic?

The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs. Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p. However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.     

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitroresponses of pulmonary arteries (6-mm OD) from the dorsocaudal (top)and cranioventral (bottom) lung regions to endothelium-dependentvasodilators (methacholine, bradykinin, and calcium ionophore A-23187).Methacholine relaxed endothelium-intact top vessels; however, in bottomvessels, a small relaxation preceded a profound contraction. In topvessels, removal of endothelial cells converted relaxation tocontraction, and in bottom vessels it abolished relaxation and enhancedcontraction. Bradykinin and A-23187 were more potent and caused greaterendothelium-mediated relaxation in top than in bottom arteries. Theendothelium-independent vasodilator sodium nitroprusside caused similarrelaxations in all rings.N-nitro-L-arginine andN^G-monomethyl-L-arginine andmethylene blue abolished relaxation of top and bottom arteries tomethacholine; meclofenamate had little effect. We conclude thatregional differences in endothelium-mediated relaxation are caused bydifferences in the magnitude of the endothelial release of nitricoxide. Similar differences in endothelium-dependent flow-mediatedvasodilation and endothelial nitric oxide release may result inpreferential perfusion of caudodorsal lung regions.

     

Enzymes in jasmonate biosynthesis – Structure,function, regulation

Jasmonates are a growing class of lipid-derived signaling molecules with diverse functions ranging from the initiation of biotic and abiotic stress responses to the regulation of plant growth and development. Jasmonate biosynthesis originates from polyunsaturated fatty acids in chloroplast membranes. In a first lipoxygenase-catalyzed reaction molecular oxygen is introduced to yield their 13-hydroperoxy derivatives. These fatty acid hydroperoxides are converted by allene oxide synthase and allene oxide cyclase to 12-oxophytodienoic acid (OPDA) and dinor-OPDA, i.e. the first cyclic intermediates of the pathway. In the subsequent step, the characteristic cyclopentanone ring structure of jasmonates is established by OPDA reductase. Until recently, jasmonic acid has been viewed as the end product of the pathway and as the bioactive hormone. It becomes increasingly clear, however, that biological activity extends to and may even differ between the various jasmonic acid metabolites and conjugates as well as its biosynthetic precursors. It has also become clear that oxygenated fatty acids give rise to a vast variety of bioactive compounds including but not limited to jasmonates. Recent insights into the structure, function, and regulation of the enzymes involved in jasmonate biosynthesis help to explain how this variety is generated while specificity is maintained.     

What is the role of PACAP in gonadotrope function?

Strong evidence in favor of a direct action of hypothalamic PACAP at the pituitary to modulate gonadotrope function has been acquired mainly by in vitro studies using cultured pituitary cells or gonadotrope cell lines. In particular, PACAP has been shown to cooperate with GnRH, the primary regulator of gonadotropes, to regulate/modulate gonadotropin subunit gene expression, gonadotropin release as well as gonadotrope responsiveness. These effects of PACAP appear to be due essentially to its high potent stimulatory action on the cAMP/protein kinase pathway. Ensuing mechanisms include signaling cross-talk and/or enhanced gene expression within gonadotropes. PACAP may also indirectly operate on these cells through paracrine mechanisms. While PACAP has long been viewed as a hypophysiotropic factor, a locally produced PACAP has also been described. Interestingly, both appear similarly up-regulated at proestrus of the reproductive cycle in female rats. Further in vivo investigation is now necessary to ascertain the physiological relevance of the observed pituitary PACAP effects and especially to evaluate the respective contribution of hypothalamic and pituitary PACAP in the dynamic control of gonadotrope function.     

Does adiposity affect muscle function during walking in children?

The biomechanical mechanisms responsible for the altered gait in obese children are not well understood, particularly as they relate to increases in adipose tissue. The purpose of this study was to test the hypotheses that as body-fat percentage (BF%) increased: (1) knee flexion during stance would decrease while pelvic obliquity would increase; (2) peak muscle forces normalized to lean-weight would increase for gluteus medius, gastrocnemius, and soleus, but decrease for the vasti; and (3) the individual muscle contributions to center of mass (COM) acceleration in the direction of their primary function(s) would not change for gluteus medius, gastrocnemius, and soleus, but decrease for the vasti. We scaled a musculoskeletal model to the anthropometrics of each participant (n=14, 8–12 years old, BF%: 16–41%) and estimated individual muscle forces and their contributions to COM acceleration. BF% was correlated with average knee flexion angle during stance (r=−0.54, p=0.024) and pelvic obliquity range of motion (r=0.78, p<0.001), as well as with relative vasti (r=−0.60, p=0.023), gluteus medius (r=0.65, p=0.012) and soleus (r=0.59, p=0.026) force production. Contributions to COM acceleration from the vasti were negatively correlated to BF% (vertical— r=−0.75, p=0.002, posterior— r=−0.68, p=0.008), but there were no correlation between BF% and COM accelerations produced by the gastrocnemius, soleus and gluteus medius. Therefore, we accept our first, partially accept our second, and accept our third hypotheses. The functional demands and relative force requirements of the hip abductors during walking in pediatric obesity may contribute to altered gait kinematics.     

Adipose tissue derived-factors impaired pancreatic β-cell function in diabetes

Inflammatory factors produced and secreted by adipose tissue, in particular peri-pancreatic adipose tissue (P-WAT), may influence pancreatic β-cell dysfunction. Using the ZDF Rat model of diabetes, we show the presence of infiltrating macrophage (ED1 staining) on pancreatic tissue and P-WAT in the pre-diabetes stage of the disease. Then, when the T2D is installed, infiltrating cells decreased. Meanwhile, the P-WAT conditioned-medium composition, in terms of inflammatory factors, varies during the onset of the T2D. Using chemiarray technology, we observed an over expression of CXCL-1, -2, -3, CCL-3/MIP-1α and CXCL-5/LIX and TIMP-1 in the 9?weeks old obese ZDF pre-diabetic rat model. Surprisingly, the expression profile of these factors decreased when animals become diabetic (12?weeks obese ZDF rats). The expression of these inflammatory proteins is highly associated with inflammatory infiltrate. P-WAT conditioned-medium from pre-diabetes rats stimulates insulin secretion, cellular proliferation and apoptosis of INS-1 cells. However, inhibition of conditioned-medium chemokines acting via CXCR2 receptor do not change cellular proliferation apoptosis and insulin secretion of INS-1 cells induced by P-WAT conditioned-medium. Taken together, these results show that among the secreted chemokines, increased expression of CXCL-1, -2, -3 and CXCL-5/LIX in P-WAT conditioned-medium is concomitant with the onset of the T2D but do not exerted a direct effect on pancreatic β-cell dysfunction.     

Evidence for conserved function of γ-glutamyltranspeptidase in Helicobacter genus

The confounding consequences of Helicobacter bilis infection in experimental mice populations are well recognized, but the role of this bacterium in human diseases is less known. Limited data are available on virulence determinants of this species. In Helicobacter pylori, γ-glutamyltranspeptidase (γGT) contributes to the colonization of the gastric mucosa and to the pathogenesis of peptic ulcer. The role of γGT in H. bilis infections remains unknown. The annotated genome sequence of H. bilis revealed two putative ggt genes and our aim was to characterize these H. bilis γGT paralogues. We performed a phylogenetic analysis to understand the evolution of Helicobacter γGTs and to predict functional activities of these two genes. In addition, both copies of H. bilis γGTs were expressed as recombinant proteins and their biochemical characteristics were analysed. Functional complementation of Esherichia coli deficient in γGT activity and deletion of γGT in H. bilis were performed. Finally, the inhibitory effect of T-cell and gastric cell proliferation by H. bilis γGT was assessed. Our results indicated that one gene is responsible for γGT activity, while the other showed no γGT activity due to lack of autoprocessing. Although both H. bilis and H. pylori γGTs exhibited a similar affinity to L-Glutamine and γ-Glutamyl-p-nitroanilide, the H. bilis γGT was significantly less active. Nevertheless, H. bilis γGT inhibited T-cell proliferation at a similar level to that observed for H. pylori. Finally, we showed a similar suppressive influence of both H. bilis and H. pylori γGTs on AGS cell proliferation mediated by an apoptosis-independent mechanism. Our data suggest a conserved function of γGT in the Helicobacter genus. Since γGT is present only in a few enterohepatic Helicobacter species, its expression appears not to be essential for colonization of the lower gastrointestinal tract, but it could provide metabolic advantages in colonization capability of different niches.     

Are TRP channels involved in sperm development and function?

Spermatozoa must translate information from their environment and the egg to achieve fertilization in sexually reproducing animals. These tasks require decoding a variety of signals in the form of intracellular Ca(2+) changes. As TRP channels constitute a large family of versatile multi-signal transducers, they are interesting subjects in which to explore their possible participation in sperm function. Here, we review the evidence for their presence and involvement in sperm motility, maturation, and the acrosome reaction, an exocytotic process required for sperm-egg fusion. Since store-operated Ca(2+) entry (SOCE) has been proposed to play an important role in these three functions, the main proteins responsible for this transport (STIM and ORAI) and their interaction with TRPs are also discussed. Improving our tools to solve infertility, improve animal breeding, and preserve biodiversity requires a better understanding of how Ca(2+) is regulated in spermatozoa.     

Rhesus factors and ammonium: a function in efflux?

Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters.     

Regional structure–function relationships in mouse aortic valve tissue

Site-specific biomechanical properties of the aortic valve play an important role in native valve function, and alterations in these properties may reflect mechanisms of degeneration and disease. Small animals such as targeted mutagenesis mice provide a powerful approach to model human valve disease pathogenesis; however, physical mechanical testing in small animals is limited by valve tissue size. Aortic valves are comprised of highly organized extracellular matrix compartmentalized in cusp and annulus regions, which have different functions. The objective of this study was to measure regional mechanical properties of mouse aortic valve tissue using a modified micropipette aspiration technique. Aortic valves were isolated from juvenile, adult and aged adult C57BL/6 wild type mice. Tissue tensile stiffness was determined for annulus and cusp regions using a half-space punch model. Stiffness for the annulus region was significantly higher compared to the cusp region at all stages. Further, aged adult valve tissue had decreased stiffness in both the cusp and annulus. Quantitative histochemical analysis revealed a collagen-rich annulus and a proteoglycan-rich cusp at all stages. In aged adult valves, there was proteoglycan infiltration of the annulus hinge, consistent with the observed mechanical differences over time. These findings indicate that valve tissue biomechanical properties vary in wild type mice in a region-specific and age-related manner. The micropipette aspiration technique provides a promising approach for studies of valve structure and function in small animal models, such as transgenic mouse models of valve disease.     

α-Crystallin chaperone function in diabetic rat and human lenses

This study focussed on the effect of diabetes on the chaperone function of alpha-crystallin. The authors relied on diabetic rats with a wide range of plasma glucose levels and non-diabetic control rats to establish a possible relationship between severity of diabetes and alpha-crystallin chaperone activity. In addition, 52-56 and 63-69 year-old diabetic and non-diabetic human lenses were used to show whether diabetes affects alpha-crystallin chaperone activity in human lenses. Correlation between plasma glucose levels and loss of chaperone activity of the alphaL-crystallin fraction in diabetic rats indicated good correlation. The glycemic threshold, reported before for cataract development in diabetic rats, seems to be valid for the chaperone activity loss as well. Analysis of the human lens alphaL-crystallin showed lower chaperone activity in all the diabetic lenses than in the age-matched control lenses. In the 63-69 age group, the loss in chaperone activity due to diabetes was significantly larger than in the 52-56 age group suggesting a dominant effect of duration of diabetes.