Structure and inhibition analysis of the mouse SAD-B C-terminal fragment

The SAD (synapses of amphids defective) kinases, including SAD-A and SAD-B, play important roles in the regulation of neuronal development, cell cycle, and energy metabolism. Our recent study of mouse SAD-A identified a unique autoinhibitory sequence (AIS), which binds at the junction of the kinase domain (KD) and the ubiquitin-associated (UBA) domain and exerts autoregulation in cooperation with UBA. Here, we report the crystal structure of the mouse SAD-B C-terminal fragment including the AIS and the kinase-associated domain 1 (KA1) at 2.8 Å resolution. The KA1 domain is structurally conserved, while the isolated AIS sequence is highly flexible and solvent-accessible. Our biochemical studies indicated that the SAD-B AIS exerts the same autoinhibitory role as that in SAD-A. We believe that the flexible isolated AIS sequence is readily available for interaction with KD-UBA and thus inhibits SAD-B activity.     

SAD-A Potentiates Glucose-Stimulated Insulin Secretion as a Mediator of Glucagon-Like Peptide 1 Response in Pancreatic β Cells

Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic β cells, which can be restored by glucagon-like peptide 1 (GLP-1), an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet β cells remain poorly understood. Here we investigated a role of SAD-A, an AMP-activated protein kinase (AMPK)-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretin''s effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS and further potentiated GLP-1''s effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is expressed exclusively in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through cyclic AMP (cAMP)/Ca²⁺-dependent signaling pathways in islet β cells. Furthermore, we identified Thr443 as a key autoinhibitory phosphorylation site which mediates SAD-A''s effect on incretin response in islet β cells. Consequently, ablation of Thr443 significantly enhanced GLP-1''s effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet β cells.     

DFLP,SSCP, and SSR markers for Δ9-stearoyl-acyl carrier protein desaturases strongly expressed in developing seeds of sunflower: intron lengths are polymorphic among elite inbred lines

Stearoyl-acyl carrier protein desaturase (SAD, EC 1.14.99.6) produces oleic acid (18:1 9) by desaturating 18:0. SAD genes have been targets for breeding and engineering oilseed crops with increased stearic acid (18:0). Our aim was to clone, describe, and develop genetic markers for the SAD genes of sunflower (Helianthus annuus L.). Nineteen SAD cDNA clones were partially sequenced and found to belong to two groups. Full-length cDNAs from each group (SAD6 and SAD17) were completely sequenced. The amino acid identity of SAD6 and SAD17 was 89%. Both genes were strongly expressed in developing seeds, moderately expressed in leaves and flowers, and weakly expressed in cotyledons, roots, and stems. One intron was found in SAD6 and two introns were found in SAD17. The SAD introns from two inbred lines (HA370 and HA372) were sequenced and found to vary in length and nucleotide sequence. The length variants were caused by monomeric repeat length differences, insertions, and deletions. Three long poly-T repeats (T₉ to T₃₉) were found in one of the SAD17 introns. Three short adjacent CA repeats were found in the 5-untranslated region of SAD6. DNA fragment length polymorphism (DFLP), single-strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for SAD6 and SAD17 by developing primers to flank introns or the CA repeats. Two of six DFLP, four of six SSCP, and one of two SSR markers were polymorphic among eight elite inbred lines. The polymorphic information contents for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Most of the polymorphisms were caused by intron fragment length polymorphisms. Introns may be an excellent source of hypervariable markers in sunflower and other crop plants.     

Mutation of SAD2, an importin beta-domain protein in Arabidopsis, alters abscisic acid sensitivity

A number of protein and RNA-processing mutants have been shown to affect ABA sensitivity. A new mutant, sad2-1, was isolated from a T-DNA mutagenized population of RD29A:LUC plants and shown to have increased luminescence after ABA, salt, cold or polyethylene glycol treatments. Expression of several ABA- and stress-responsive genes was higher in the mutant than in the wild type. sad2-1 also exhibited ABA hypersensitivity in seed germination and seedling growth. SAD2 was found to encode an importin beta-domain family protein likely to be involved in nuclear transport. SAD2 was expressed at a low level in all tissues examined except flowers, but SAD2 expression was not inducible by ABA or stress. Subcellular localization of GFP-tagged SAD2 showed a predominantly nuclear localization, consistent with a role for SAD2 in nuclear transport. Knockout of the closest importin beta homolog of SAD2 in Arabidopsis did not duplicate the sad2 phenotype, indicating that SAD2 plays a specific role in ABA signaling. Analysis of RD29A:LUC luminescence and ABA and stress sensitivity in double mutants of sad2-1 and sad1 or abh1-7, a newly isolated allele of ABH1 also in the RD29A:LUC background, suggested that SAD2 acts upstream of or has additive effects with these two genes. The results suggest a role for nuclear transport in ABA signal transduction, and the possible roles of SAD2 in relation to that of SAD1 and ABH1 are discussed.     

SAD,a stearoyl-acyl carrier protein desaturase highly expressed in high-oil maize inbred lines

As special maize with more than 6% oil concentration in the grain, high-oil maize has received increased interest recently. To date, little is known about the expressions of genes involved in fatty acid metabolism of high-oil maize. Stearoyl-acyl carrier protein desaturase (SAD) is a key enzyme that converts stearic acid to oleic acid. In this study, two-dimensional electrophoresis, gas chromatography, and real-time PCR were used to determine the expressions of SAD at three seed development stages in high-oil and normal maize inbred lines. SAD was significantly more abundantly expressed in high-oil maize than in normal maize, not only at the protein and mRNA levels, but also at the product level. These results suggested that a high expression of SAD may play an important role in increasing oil concentration in high-oil maize.     

SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.     

The genetic basis of pollinator adaptation in a sexually deceptive orchid

In plants, pollinator adaptation is considered to be a major driving force for floral diversification and speciation. However, the genetic basis of pollinator adaptation is poorly understood. The orchid genus Ophrys mimics its pollinators' mating signals and is pollinated by male insects during mating attempts. In many species of this genus, chemical mimicry of the pollinators' pheromones, especially of alkenes with different double-bond positions, plays a key role for specific pollinator attraction. Thus, different alkenes produced in different species are probably a consequence of pollinator adaptation. In this study, we identify genes that are likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturases (SAD), in three closely related Ophrys species, O. garganica, O. sphegodes, and O. exaltata. Combining floral odor and gene expression analyses, two SAD homologs (SAD1/2) showed significant association with the production of (Z)-9- and (Z)-12-alkenes that were abundant in O. garganica and O. sphegodes, supporting previous biochemical data. In contrast, two other newly identified homologs (SAD5/6) were significantly associated with (Z)-7-alkenes that were highly abundant only in O. exaltata. Both molecular evolutionary analyses and pollinator preference tests suggest that the alkenes associated with SAD1/2 and SAD5/6 are under pollinator-mediated divergent selection among species. The expression patterns of these genes in F(1) hybrids indicate that species-specific expression differences in SAD1/2 are likely due to cis-regulation, while changes in SAD5/6 are likely due to trans-regulation. Taken together, we report a genetic mechanism for pollinator-mediated divergent selection that drives adaptive changes in floral alkene biosynthesis involved in reproductive isolation among Ophrys species.     

紫苏硬脂酰-ACP脱氢酶基因家族鉴定及表达分析北大核心CSCD

硬脂酰-ACP脱氢酶(SAD)催化硬脂酸脱氢生成油酸,是形成不饱和脂肪酸的关键酶。该研究从紫苏转录组数据库中筛选鉴定紫苏硬脂酰-ACP脱氢酶(PfSAD)家族基因,并进行生物信息学分析及保守功能域分析,用qRT-PCR技术检测PfSADs各成员在不同组织中的表达特性,以探讨PfSAD家族基因在调控种子脂肪酸组分中的作用,为紫苏脂肪酸组分的遗传改良提供基因元件。结果显示:(1)从该课题组前期自测的紫苏转录组数据库中共检测出6个PfSAD家族基因,其编码蛋白的氨基酸长度介于367~396 aa之间,均具有SAD的保守结构域和二铁中心,预测其基因编码蛋白均定位于叶绿体。(2)多序列比对结果显示,紫苏PfSADs蛋白序列与拟南芥、蓖麻及可可等植物的SAD蛋白序列相似性均在50%以上;系统进化分析显示,6个紫苏SAD蛋白被分为3个亚组,其中第一个亚组包含PfSAD1,第二亚组包含PfSAD2、PfSAD3,第三亚组包含PfSAD4、PfSAD5和PfSAD6。(3)实时荧光定量PCR分析发现,PfSADs各成员在‘晋紫苏1号’不同组织中的表达量差异显著,其中PfSAD1主要在叶中表达,PfSAD2、PfSAD3、PfSAD4和PfSAD5在种子中表达量较高,PfSAD6在花中具有显著表达优势。研究表明,PfSADs具有典型的保守基序及催化SAD的活性中心,其各成员在不同的组织中高表达,推测这6个基因均参与了硬脂酰ACP(C18∶0-ACP)脱氢生成油酰基ACP(Δ9C18∶1-ACP)的过程,在紫苏油脂合成代谢过程中发挥重要作用。     

SAD-A and AMPK kinases: The “yin and yang” regulators of mTORC1 signaling in pancreatic β cells

SAD-A kinase is a member of the AMPK-related family of kinases, which are under the control of LKB1 kinase. In the human kinome, SAD-A is most closely related to AMPK, a key energy sensor and master regulator of metabolism. In contrast to AMPK, little is known about the physiological function of the SAD-A kinase in metabolism. Recent studies using knockout mice have revealed a striking role of the SAD-A kinase in regulating dynamic functions of islet β cells, from glucose-stimulated insulin secretion (GSIS), islet β-cell size and mass, to GLP-1 response as the first tissue-specific effector of mTORC1 signaling. These studies suggest that SAD-A and AMPK kinase may function as the positive and negative regulators of mTORC1 signaling in islet β cells. Importantly, these findings have implicated SAD-A kinase as a novel drug target for the treatment of type 2 diabetes.