Structure and function of WD40 domain proteins

The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.     

Proteomic analysis of the hepatic tissue of Long-Evans Cinnamon (LEC) rats according to the natural course of Wilson disease

Copper-induced toxicity is important in the pathogenic process of Wilson's disease (WD). Using Long-Evans Cinnamon (LEC) rats, an animal model of WD, the study was undertaken to identify proteins involved in the process of WD and to investigate their functional roles in copper-induced hepatotoxicity. In early stages, expression levels of mitochondrial matrix proteins including agmatinase, isovaleryl coenzyme A dehydrogenase, and cytochrome b5 were downregulated. As mitochondrial injuries progressed, along with subsequent apoptotic processes, expressions of malate dehydrogenase 1, annexin A5, transferrin, S-adenosylhomocysteine hydrolase, and sulfite oxidase 1 were differentially regulated. Notably, the expression of malate dehydrogenase 1 was downregulated while the annexin A5 was overexpressed in an age-dependent manner, indicating that these proteins may be involved in the WD process. In addition, pronounced under-expression of S-adenosylhomocysteine hydrolase in elderly LEC rats, also involved in monoamine neurotransmitter metabolism, indicates that this protein might be related to the development of neurological manifestations in WD. The results of our study help to understand the pathogenic process of WD in hepatic tissues, identifying the important proteins associated with the disease process of WD, and to investigate the molecular pathogenic process underlying the development of neurological manifestations in WD.     

川西亚高山森林木质残体及其附生苔藓持水特性

木质残体及其附生苔藓是森林生态系统的基本结构,在水源涵养、水土保持、生物多样性保育、碳和养分循环等方面具有重要作用。然而,有关亚高山森林木质残体及其附生苔藓的水源涵养功能研究尚未见报道。因此,以川西亚高山针叶林区紫果云杉原始林、岷江冷杉原始林、方枝柏原始林等8种典型森林类型为研究对象,调查研究了木质残体及其附生苔藓持水能力随林型、腐烂等级和径级的变化特征。结果表明：（1）亚高山针叶林木质残体饱和储水量在林型之间变化显著。其中,紫果云杉原始林具有最大的饱和储水量（22.06 mm）,柳树次生林木质残体饱和储水量最小（4.55 mm）,但林型之间木质残体饱和持水率的差异不显著。（2）不同林型的附生苔藓持水能力具有显著差异。其中,紫果云杉原始林木质残体附生苔藓饱和储水量最高（7.01 mm）,方枝柏原始林最低（0.21 mm）。（3）腐烂等级显著影响木质残体饱和储水量,IV腐烂等级的饱和储水量较高（3.77 mm）,II腐烂等级较低（0.75 mm）;木质残体饱和持水率和附生苔藓饱和储水量与腐解程度显著正相关,均符合Q（Q''）=ex²+fx+g的函数关系式。（4）木质残体和附生苔藓的饱和储水量均随径级增大而升高,大径级（D5）的饱和储水量占比分别超过60%和40%。可见,林型和木质残体腐解程度及径级大小是决定亚高山针叶林区木质残体及其附生苔藓持水性能的关键因子,特别是附生苔藓对于提高亚高山针叶林水源涵养具有重要作用。     

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

Differences in performance between trained and untrained subjects during a 30-s sprint test in a wheelchair ergometer

To compare physiological responses and propulsion technique of able bodied subjects with no prior experience of wheelchairs (AB) and wheelchair dependent subjects (WD), ten AB and nine WD performed a 30-s sprint test in a wheelchair ergometer. The WD had spinal cord injuries with a lesion at T8 or lower. The WD and AB did not show significantly different physiological responses. The power values averaged for the right wheel over the 30 s of the test were 50.2 (SD 14.7) W and 48.0 (SD 4.4) W for WD and AB, respectively. No significant differences in torque application could be discerned, although WD subjects seemed to have a more flattened torque curve with a smaller negative deflection at the beginning of the push. The WD applied a significantly higher horizontal propulsive force to the handrims but did not apply force more effectively. The percentages of effective force to total propulsive force were 61 (SD 16)% for WD and 57 (SD 4)% for AB. With regard to the kinematic parameters, AB followed the handrims significantly longer than WD (end angle AB 65°, WD 44°), started the push phase with their arms more in retroflexion and flexed their trunks further forward. The AB did however show a movement pattern comparable to that of wheelchair athletes measured in a comparable experiment. It could not be decided conclusively that inexperience in wheelchair propulsion led to a less effective propulsion technique. Despite the selection of WD with respect to lesion level, interindividual differences in terms of level of training may have been responsible for the absence of significant results.     

Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis

ATP7B mutations result in Cu storage in the liver and brain in Wilson disease (WD). Atox1 and COMMD1 were found to interact with ATP7B and involved in copper transport in the hepatocyte. To understand the molecular etiology of WD, we analyzed ATP7B, Atox1 and COMMD1 genes. Direct sequencing of (i) ATP7B gene was performed in 112 WD patients to identify the spectrum of disease-causing mutations in the French population, (ii) Atox1 gene was performed to study the known polymorphism 5'UTR-99T>C in 78 WD patients with two ATP7B mutations and (iii) COMMD1 gene was performed to detect the nucleotide change c.492GAT>GAC. MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was performed in WD patients presenting only one ATP7B mutation. Among our 112 WD unrelated patients, 83 different ATP7B gene mutations were identified, 27 of which were novel. Two ATP7B mutations were identified in 98 WD cases, and one mutation was identified in 14 cases. In two of these 14 WD patients, we identified the deletion of exon 4 of the ATP7B gene by MLPA technique. In 78 selected patients of the cohort with two mutations in ATP7B, we have examined genotype-phenotype correlation between the detected changes in Atox1 and COMMD1 genes, and the presentation of the WD patients. Based on the data of this study, no major role can be attributed to Atox1 and COMMD in the pathophysiology or clinical variation of WD.     

Tree growth traits and social status affect the wood density of pioneer species in secondary subtropical forest

Wood density (WD) is not only an important parameter to estimate aboveground biomass but also an indicator of timber quality and plant adaptation strategies to stressful conditions (i.e., windthrow, pests, and pathogens). This study had three objectives: (1) to compare WD among seven subtropical tree species; (2) to determine how tree growth traits may influence possible differences in WD between the pioneer and shade‐tolerant species; and (3) to examine whether or not WD differs by tree social status (dominant vs. suppressed trees) within species. To do this, 70 trees were destructively harvested. From each tree, disks at different stem heights were obtained and subjected to a method of stem analysis to measure whole tree level WD. The results showed that WD differed significantly among the seven species (p < .001). Their average WD was 0.537 g/cm³, ranging from 0.409 g/cm³ for Choerospondias axillaris to 0.691 g/cm³ for Cyclobalanopsis glauca. The average WD of the four pioneer species (0.497 ± 0.13 g/cm³) was significantly lower (p < .01) than that of the three shade‐tolerant species (0.589 ± 0.12 g/cm³). The WD of the pioneers had a significant positive correlation with their stem diameter at breast height (DBH), tree height (H), and tree age, but WD had a significant negative correlation with relative growth rate (RGR). In contrast, the WD of the shade‐tolerant tree species had no significant relationships with DBH, H, tree age, or RGR. The dominant trees of the pioneer species had a higher WD than the suppressed trees, whereas the shade‐tolerant species had a lower WD for dominant trees than the suppressed trees. However, the differences in WD between dominant and suppressed trees were not significant. Taken together, the results suggest that classifying species into pioneer and shade‐tolerant groups to examine the effects of tree growth traits and social status could improve our understanding of intra‐ and interspecific variation in WD among subtropical tree species.     

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis,Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.     

稻瘟菌WD重复功能域中SSR的变异及其对蛋白结构的影响

含WD重复功能域的蛋白能够参与信号传导、转录调控、RNA剪切、细胞的凋亡等多种功能,在病原菌与寄主植物蛋白互作的过程中扮演着重要的角色。本研究分析了稻瘟病菌基因组中94个WD功能域基因编码区和调控区中SSR的组成、分布,并检测了7个蛋白编码区中SSR的变异及其对蛋白二级结构的影响。结果表明,WD功能域基因的编码区和调控区中都含有大量的SSR,但是SSR在这些基因的外显子区、内含子区、5’一UTR和3’一UTR区中SSR的组成和分布均不相同;编码区中三碱基和六碱基SSR分布较多,这些SSR基序大都表现为GC含量较高和其所编码的亲水性氨基酸出现的频率远远高于疏水性氨基酸的特点。且检测的7个WD功能域基因的编码区中的SSR位点均具有丰富的多态性,通过Antheprot（DPM）软件预测发现：SSR的变异对蛋白的二级结构有一定影响。这暗示着SSR的变异对致病相关基因的变异起着十分重要的作用。     

Relationships between nerves and myofibroblasts during cutaneous wound healing in the developing rat

Contraction of skin excision wounds is affected by age and the presence of peripheral nerves. The present study examined relationships between peripheral innervation, wound contractile cells, and rate of wound closure to determine whether these are altered during development. Full-thickness 4-mm-diameter circular flaps were excised from the interscapular skin of rats on postnatal day (PND) 5, PND 12, or PND 60. Wounds of PND 5 and PND 12 rats contracted 45% between post-wound days (WD) 3 and 5 and more slowly thereafter, with a scar 9-14% of the original wound size by WD 21. In contrast, PND 60 wounds contracted only 22% between WD 3 and 5, and the residual scar at WD 21 was 40% of the original wound size. In younger rats, alpha-smooth muscle actin-immunoreactive myofibroblasts first appeared on WD 2 and attained maximum density at WD 5. Innervation, as assessed by protein gene product 9.5 immunoreactivity, appeared by WD 3 and increased rapidly through WD 7 in younger rats. In PND 60 wounds, myofibroblasts did not appear until WD 5 and did not attain a maximum until day 10. Nerve ingrowth was not significant until WD 10 and was depressed relative to younger rats throughout the healing phase. Wound nerves were predominantly immunoreactive to calcitonin gene-related peptide, and synaptophysin-immunostaining revealed close associations between varicosities and myofibroblasts. These findings suggest that wound myofibroblasts may be a target of peripheral nerves, and delayed wound closure in mature rats is associated with deficiencies in both myofibroblasts and innervation.     

The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1

Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cepsilon (PKCepsilon) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na(+)/H(+) exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1-PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC(50) values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1-NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel beta-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a beta-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCepsilon binding site on the WD6 repeat of RACK1.     

The C-terminal WD40 repeats on the TOPLESS co-repressor function as a protein–protein interaction surface

Plant Molecular Biology - The two predicted WD40 propellers on TOPLESS function as protein–protein interaction domains. The 1st WD40 propeller mediates interaction with RAV1, and the 2nd WD40...     

Response of barley seedlings to water deficit and enhanced UV-B irradiation acting alone and in combination

Responses of barley seedlings to water deficit (WD) induced by polyethylene glycol (PEG 6000) and ultraviolet (UV-B; 280–320 nm) radiation and their interaction (UV-B + WD) were examined. A decrease in dry matter yield and water content of leaves and roots was observed following application of WD and UV-B + WD, while no changes were found after treating barley plants with UV-B. Proline content was increased in leaves under WD conditions and UV-B + WD. In contrast, UV-B treatment had no effect on the accumulation of proline in leaves of barley plants. Changes in root proline content showed a varied response: WD induced an increase in the level of this amino acid, while UV-B as well as UV-B + WD suppressed root proline content. The lipid peroxidation product malondialdehyde (MDA) was increased in leaves under WD and UV-B + WD stresses. Root MDA content increased in WD-stressed plants, but it decreased in the case of combined application of both stresses. The applied stress factors operated in a variable manner on phenylpropanoid metabolism. Phenylalanine ammonia-lyase (PAL) activity in leaves and roots was stimulated after exposure to WD and application of UV-B + WD stresses, while UV-B stress did not affect its activity. On the other hand, UV-B treatment enhanced the activity of 4:coumarate-CoA ligase (4CL) in leaves and this enhancement was positively correlated with the accumulation of anthocyanins and flavonols. However, the combined application of WD and UV-B reduced the positive effect of UV-B on the accumulation of these compounds and the activity of 4CL. Surprisingly, anthocyanins and flavonols were not detected in roots of examined barley seedlings despite increased 4CL activity. The results suggest that UV-B-induced activation of 4CL as well as accumulation of anthocyanin and flavonols in leaves is beneficial for the response to this stress factor. On the other hand, WD-induced reduction of the effect of UV-B on 4CL activity and the contents of anthocyanin and flavonol might be a cause of membrane damage in UV-B- and WD-stressed plants. In addition, conversely to what could be expected, the UV-B effect was perceived by the water-stressed roots, which exhibited reduced lipid peroxidation (MDA) and proline accumulation in WD-stressed plants exposed to UV-B.     

Analysis of most common mutations R778G, R778L, R778W, I1102T and H1069Q in Indian Wilson disease patients: Correlation between genotype/phenotype/copper ATPase activity

The present study was intented to estimate the frequencies of the most common mutations (R778L, R778W, R778G, I1102T and H1069Q) of ATP7B in Indian Wilson disease (WD) population and to explore the correlation between genotype/phenotype and copper ATPase activity. A total of 33 WD patients and their family members from North West states of India were examined. The H1069Q, R778W and R778L mutations were absent in these WD patients. R778W and I1102T mutations were present in 36% of WD patients. Family analysis for these mutations using PCR-RFLP documented 5 carriers and 2 asymptomatic WD patients. The copper ATPase activity in WD patients was significantly reduced (50%) than that of control individuals. No significant difference was observed in copper stimulated ATPase activity between homozygous (R778W/R778W, I1102T/I1102T) and compound heterozygous (R778W/unknown mutation, I1102T/unknown mutation) WD patients. Serum ceruloplasmin, serum copper levels were significantly lower in homozygous WD patients than that of compound heterozygous. However, no significant difference was observed in liver copper contents between heterozygous and homozygous patients. In conclusion, the data suggest that R778W and I1102T are most common mutations and provide the basis of genetic (PCR-RFLP) diagnostic tool for Indian WD patients as well as in siblings/parents where biochemical parameters are ambiguous.     

Changes in the Non-Photochemical Quenching of Chlorophyll Fluorescence During Aging of Wheat Flag Leaves

Ultrastructural changes in chloroplasts of primary leaves of 15-d-old bean plants (Phaseolus vulgaris L. cv. Cheren Starozagorski) in response to a single stress (increasing water deficit, WD) as well as to combined stress (WD plus high temperature, WD+HT) were investigated under the possible protective or reparatory effects of the carbamide cytokinin 4-PU-30 [N-(2-chloro-4-pyridyl)-N-phenylurea] applied before or after the stress. Essential structural changes in chloroplast ultrastructure occurred mainly in plants that had experienced WD+HT: the thylakoids were swollen, the envelope was destroyed, and the spatial orientation of inner membrane system was not typical. Changed starch accumulation was also observed. 4-PU-30 protected chloroplast ultrastructure under WD+HT.     

Comparative hydraulic architecture of tropical tree species representing a range of successional stages and wood density

Plant hydraulic architecture (PHA) has been linked to water transport sufficiency, photosynthetic rates, growth form and attendant carbon allocation. Despite its influence on traits central to conferring an overall competitive advantage in a given environment, few studies have examined whether key aspects of PHA are indicative of successional stage, especially within mature individuals. While it is well established that wood density (WD) tends to be lower in early versus late successional tree species, and that WD can influence other aspects of PHA, the interaction of WD, successional stage and the consequent implications for PHA have not been sufficiently explored. Here, we studied differences in PHA at the scales of wood anatomy to whole-tree hydraulic conductance in species in early versus late successional Panamanian tropical forests. Although the trunk WD was indistinguishable between the successional groups, the branch WD was lower in the early successional species. Across all species, WD correlated negatively with vessel diameter and positively with vessel packing density. The ratio of branch:trunk vessel diameter, branch sap flux and whole-tree leaf-specific conductance scaled negatively with branch WD across species. Pioneer species showed greater sap flux in branches than in trunks and a greater leaf-specific hydraulic conductance, suggesting that pioneer species can move greater quantities of water at a given tension gradient. In combination with the greater water storage capacitance associated with lower WD, these results suggest these pioneer species can save on the carbon expenditure needed to build safer xylem and instead allow more carbon to be allocated to rapid growth.     

Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP × PS1 knock-in mice

A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP × PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-β (Aβ) pathogenesis. In this study, we placed 1-month-old APP × PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP × PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Aβ levels. Altered adipokine levels were also observed in APP × PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP × PS1 knock-in mice, with increased oxidative stress preceding alterations in Aβ. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.     

A novel approach to oral apoA-I mimetic therapy

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR^−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.