Integrative structure and function of the yeast exocyst complex

Exocyst is an evolutionarily conserved hetero‐octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative‐stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride cross‐links from chemical‐crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo‐EM structure, cross‐links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo‐EM structure, except for Sec5. While not observed in the cryo‐EM map, the integrative model localizes the N‐terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low‐resolution structural data can inform biologically relevant hypotheses, even in the absence of high‐resolution data.     

A Plasmodium homolog of ER tubule-forming proteins is required for parasite virulence

Reticulon and REEP family of proteins stabilize the high curvature of endoplasmic reticulum (ER) tubules. Plasmodium berghei Yop1 (PbYop1) is a REEP5 homolog in Plasmodium. Here, we characterize its function using a gene-knockout (Pbyop1∆). Pbyop1∆ asexual stage parasites display abnormal ER architecture and an enlarged digestive vacuole. The erythrocytic cycle of Pbyop1∆ parasites is severely attenuated and the incidence of experimental cerebral malaria is significantly decreased in Pbyop1∆-infected mice. Pbyop1∆ sporozoites have reduced speed, are slower to invade host cells but give rise to equal numbers of infected HepG2 cells, as WT sporozoites. We propose that PbYOP1’s disruption may lead to defects in trafficking and secretion of a subset of proteins required for parasite development and invasion of erythrocytes. Furthermore, the maintenance of ER morphology in different parasite stages is likely to depend on different proteins.     

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide‐binding leucine‐rich repeat protein (NLR)‐encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA‐Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

肠激酶在毕赤酵母中的分泌表达优化

肠激酶 (Enterokinase,EK) 是一类特异性识别切割DDDDK序列的丝氨酸蛋白酶,作为一种工具酶广泛应用于生物医药领域。目前,EK在毕赤酵母Pichia pastoris中的表达水平较低,难以应用。本研究比较了6种不同的信号肽SP1、SP2、SP3、SP4、SP7和SP8对毕赤酵母分泌表达EK的影响。在摇瓶水平上,与α-factor信号肽相比,SP1信号肽显著提高了EK的分泌表达 (从6.8 mg/L提高至14.3 mg/L),酶活从 (2 390±212) U/mL提高至 (4 995±378) U/mL。在此基础上,通过共表达毕赤酵母内源蛋白Kex2,EK酶活提高至 (7 219±489) U/mL。另外,N端融合WLR三个氨基酸进一步提高酶活至 (15 145±920) U/mL,比酶活为 (1 174 600±53 100) U/mg。EK在毕赤酵母中的高效分泌表达为未来应用奠定了基础。     

Efficient deletion of LoxP-flanked selectable marker genes from the genome of transgenic pigs by an engineered Cre recombinase

Genetically modified (GM) pigs hold great promises for pig genetic improvement, human health and life science. When GM pigs are produced, selectable marker genes (SMGs) are usually introduced into their genomes for host cell or animal recognition. However, the SMGs that remain in GM pigs might have multiple side effects. To avoid the possible side effects caused by the SMGs, they should be removed from the genome of GM pigs before their commercialization. The Cre recombinase is commonly used to delete the LoxP sites-flanked SMGs from the genome of GM animals. Although SMG-free GM pigs have been generated by Cre-mediated recombination, more efficient and cost-effective approaches are essential for the commercialization of SMG-free GM pigs. In this article we describe the production of a recombinant Cre protein containing a cell-penetrating and a nuclear localization signal peptide in one construct. This engineered Cre enzyme can efficiently excise the LoxP-flanked SMGs in cultured fibroblasts isolated from a transgenic pig, which then can be used as nuclear donor cells to generate live SMG-free GM pigs harboring a desired transgene by somatic cell nuclear transfer. This study describes an efficient and far-less costly method for production of SMG-free GM pigs.

     

Understanding cellular and molecular mechanisms of pathogenesis of diabetic tendinopathy

There is accumulating evidence of an increased incidence of tendon disorders in people with diabetes mellitus. Diabetic tendinopathy is an important cause of chronic pain, restricted activity, and even tendon rupture in individuals. Tenocytes and tendon stem/progenitor cells (TSPCs) are the dominant cellular components associated with tendon homeostasis, maintenance, remodeling, and repair. Some previous studies have shown alterations in tenocytes and TSPCs in high glucose or diabetic conditions that might cause structural and functional variations in diabetic tendons and even accelerate the development and progression of diabetic tendinopathy. In this review, the biomechanical properties and histopathological changes in diabetic tendons are described. Then, the cellular and molecular alterations in both tenocytes and TSPCs are summarized, and the underlying mechanisms involved are also analyzed. A better understanding of the underlying cellular and molecular pathogenesis of diabetic tendinopathy would provide new insight for the exploration and development of effective therapeutics.