Spt蛋白与酿酒酵母压力抗性研究进展

Spt是一大类参与酿酒酵母转录调控的蛋白质。Spt蛋白是SAGA复合体的组成部分,该复合体与基因上游TATA框区域相互作用而发挥调控作用。根据目前的研究,约有10%的酿酒酵母基因转录受到Spt蛋白的调控,且与环境压力下上调基因的调控密切相关。Spt蛋白参与的庞大的调控网络及其复杂的调节机制是当前一个研究热点。Spt蛋白还具有转座子抑制的功能,是转座子功能调控和适应性进化意义上的重要"开关"。除此之外,一些Spt蛋白可以直接调控不饱和脂肪酸的合成,从而直接影响细胞膜重塑,对于酵母广泛抗性具有重要意义。文中从以上3个维度对迄今为止Spt蛋白的研究进展进行综述,结合前期研究工作,对于Spt蛋白通过转录调控、细胞膜转变、转座活性调节用以增强酿酒酵母抗性的潜在作用谨作展望。     

A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response

A20/AN1 zinc-finger domain-containing proteins are well characterized in animals, and their role in regulating the immune response is established. Recently, such A20/AN1 zinc-finger proteins have been reported from plants. These plant proteins are involved in stress response, but their exact molecular mechanism of action is yet to be deciphered. Sequence information available in public databases has been used to conduct a survey of A20/AN1 zinc-finger proteins across diverse organisms with a special emphasis on plants. Domain analysis provides some interesting insights into their biological function, the most important being that A20/AN1 zinc-finger proteins could represent common elements of stress response in plants and animals.     

固醇调节元件结合蛋白与脂质代谢

固醇调节元件结合蛋白(sterol regulatory element-binding proteins,SREBPs)是脊椎动物细胞脂质稳态的转录调节物,可直接激活多个参与胆固醇、脂肪酸、甘油三酯、磷脂合成和摄取,以及辅助因子NADPH等基因的表达,从而调控胆固醇及脂肪酸等脂类的代谢过程。本文综述了SREBPs转运和活化的过程,以及调节细胞脂质稳态功能的分子机制,并探讨了其在脂代谢紊乱相关疾病发生中的重要作用。     

Identification of proteins involved in osmotic stress response in Enterobacter sakazakii by proteomics

Enterobacter sakazakii is considered an opportunistic food-borne pathogen, causing rare but significant illness especially in neonates. It has been proposed that the organism is relatively resistant to osmotic and dry stress compared to other species of the Enterobacteriaceae group. To understand the mechanisms involved in osmotic stress response, 2-DE protein analysis coupled to MALDI-TOF MS was employed to investigate changes in the protein profiles of E. sakazakii cells in response to two different types of osmotic stress (physical desiccation and growth in hyperosmotic media). In total, 80 differentially expressed protein spots corresponding to 53 different protein species were identified. Affiliation of proteins to functional categories revealed that a considerable number of the differentially expressed proteins from desiccated and hyperosmotic grown samples belonged to the same functional category but were regulated in opposite directions. Our data show that the protein pattern of NaCl-grown cultures reflect more or less a general down-regulation of central metabolic pathways, whereas adaptation of (non-growing) cells in a desiccated state represents an accumulation of proteins that serve some structural or protective role. The most striking effects observed for both types of osmotic stress in E. sakazakii were a significant down-regulation of the motility apparatus and the formation of filamentous cells.     

Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.     

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.     

Identification of F-box proteins that are involved in resistance to methylmercury in Saccharomyces cerevisiae

We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that overexpression of Hrt3 and of Ylr224w rendered yeast cells resistant to methylmercury. Yeast cells that overexpressed Hrt3 and Ylr224w were barely resistant to methylmercury in the presence of a proteasome inhibitor. Our results suggest the existence of some protein(s) that enhances the toxicity of methylmercury in yeast cells and, also, that overexpression of Hrt3 or Ylr224w can confer resistance to methylmercury by enhancing the polyubiquitination of this protein(s) and its degradation in proteasomes.     

Influences of different stress models on the antioxidant status and lipid peroxidation in rat erythrocytes

The aim of this study was to investigate the influences of different stress models on the antioxidant status and lipid peroxidation (LPO) in erythrocytes of rats. Swiss-Albino female rats (3 months old) were used in this study. Rats were randomly divided into the following four groups; control group (C), cold stress group (CS), immobilization stress group (IS) and cold+immobilization stress group (CS+IS). Control group was kept in an animal laboratory (22 ±2°C). Rats in CS group were placed in cold room (5°C) for 15 min/day for 15 days. Rats in IS group were immobilized for 180 min/day for 15 days. Rats in CS+IS group were exposed to both cold and immobilization stresses for 15 days. At the end of experimental periods, the activities of glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and concentration of reduced glutathione (GSH) were measured. LPO was determined by measuring the contents of thiobarbituric acid-reactive substances (TBARS). Cu,Zn-SOD activity and TBARS concentration were increased after cold and immobilization stresses, but CAT and GSH-Px activities and GSH levels were decreased. Immobilization stress decreased the activity of G-6-PD. The activities of G-6-PD, CAT and GSH-Px, and the level of GSH were lower in CS+IS group than in the control group. Cu,Zn-SOD activity and TBARS levels were increased in CS+IS group when compared with the control group. From these findings, three stress models are thought to cause oxidative stress.     

Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis

Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca(2+) homeostasis seems to have an important role during Cd(2+) injury, but the precise assessment of Ca(2+) signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca(2+) changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca(2+) signals and caused a 40% inhibition of sarcoplasmic-ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.     

Signaling pathways involved in virulence and stress response of plant-pathogenic Fusarium species

Fungal pathogens face similar stress conditions to those affecting plants and saprotrophic fungi. Therefore, mechanisms underlying fungal response to the stress factors may be well-conserved across various taxa. Saccharomyces cerevisiae was the most researched for signal transduction pathways but many of the pathways' components were later reported for filamentous fungi as well. The most widely studied pathways are those involving the G proteins, adenylate cyclase (cAMP) and mitogen-activated protein kinases (MAPKs). Apart from these, the target-of-rapamycin (TOR), calcium/calcineurin and cell wall integrity (CWI) pathways are of significant interest when stress response is considered. All these pathways were included in this review. It seems that the TOR-received signals are transferred to the CWI pathway, secondary metabolism and virulence. Specific and non-specific cellular responses of Fusarium species, triggered by signals received from the environment, were discussed, with particular focus on stress response and pathogenicity towards the plant host.     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Emerging roles of phosphoinositide-associated membrane trafficking in plant stress responses

Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell. These barriers are formed by assembly of lipids and protein in aqueous environments. Lipids not only serve as building blocks for membrane construction, but also possess regulatory functions in cellular activities. These regulatory lipids are non-uniformly distributed in membrane systems; their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly. Phosphoinositides (PIs) are phospholipids that exert regulatory effects. In recent years, research on PIs roles in regulating plant growth, development, and responses to environmental stress is increasing. Several reviews have been published on the composition of PIs, intermolecular transferring of PIs by lipid kinases (phosphatases) or PI-PLCs, subcellular localization, and specially their functions in plant developments. Herein, we review the crucial regulatory functions of PIs in plant stress responses, with a particular focus on PIs involved in membrane trafficking.     

TGD1, -2, and -3 proteins involved in lipid trafficking form ATP-binding cassette (ABC) transporter with multiple substrate-binding proteins

Members of the ATP-binding cassette (ABC) transporter family are essential proteins in species as diverse as archaea and humans. Their domain architecture has remained relatively fixed across these species, with rare exceptions. Here, we show one exception to be the trigalactosyldiacylglycerol 1, 2, and 3 (TGD1, -2, and -3) putative lipid transporter located at the chloroplast inner envelope membrane. TGD2 was previously shown to be in a complex of >500 kDa. We demonstrate that this complex also contains TGD1 and -3 and is very stable because it cannot be broken down by gentle denaturants to form a "core" complex similar in size to standard ABC transporters. The complex was purified from Pisum sativum (pea) chloroplast envelopes by native gel electrophoresis and examined by mass spectrometry. Identified proteins besides TGD1, -2, or -3 included a potassium efflux antiporter and a TIM17/22/23 family protein, but these were shown to be in separate high molecular mass complexes. Quantification of the complex components explained the size of the complex because 8-12 copies of the substrate-binding protein (TGD2) were found per functional transporter.     

Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.     

Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.