中红侧沟茧蜂气味结合蛋白MmedOBP18的原核表达及配体结合特性

【目的】研究中红侧沟茧蜂Microplitis mediator气味结合蛋白MmedOBP18在触角中的表达定位,解析MmedOBP18重组蛋白的配体结合特性。【方法】原核表达中红侧沟茧蜂气味结合蛋白MmedOBP18;采用荧光免疫组织化学技术研究MmedOBP18在中红侧沟茧蜂雌蜂触角中的表达定位;通过荧光竞争结合实验分析MmedOBP18重组蛋白与99种候选配体的结合特性。【结果】在原核表达系统中成功表达MmedOBP18重组蛋白。荧光免疫定位结果显示MmedOBP18主要表达在触角锥形感器Ⅰ内的淋巴液中。荧光竞争结合实验结果表明,MmedOBP18重组蛋白能够与16种候选配体结合,与低挥发性植物挥发物2-十三酮、十二醛、十四酸和十一酸的结合能力最强,其解离常数K_i值分别为5.21, 6.42, 6.49和6.58μmol/L;而且MmedOBP18重组蛋白与非挥发性植物次生物质棕榈酸、棉酚、槲皮素及亚麻酸也有较强的结合能力,K_i值分别为3.86, 5.07, 5.08和6.51μmol/L;此外,MmedOBP18重组蛋白与鳞翅目昆虫性信息素组分顺-9-十四碳烯醛及顺-11-十六碳醛表现出较强的结合能力,K_i值分别为9.09和11.67μmol/L,提示该蛋白在寄主定位过程中可发挥重要的作用。【结论】中红侧沟茧蜂气味结合蛋白MmedOBP18能够结合长链低挥发和非挥发性化合物,推测其在嗅觉和味觉识别中发挥双重功能,主要参与对寄主和寄主生境的化学信息的近距离识别。     

Localization and characterization of a putative cysteine desulfurase in Chlamydia psittaci

Chlamydia psittaci is an obligate intracellular pathogen with a biphasic developmental life cycle. It is auxotrophic for a variety of essential metabolites and obtains amino acids from eukaryotic host cells. Chlamydia can develop inside host cells within chlamydial inclusions. A pathway secreting proteins from inclusions into the host cellular cytoplasm is the type III secretion system (T3SS). The T3SS is universal among several Gram-negative bacteria. Here, we show that CPSIT_0959 of C. psittaci is expressed midcycle and secreted into the infected cellular cytoplasm via the T3SS. Recombinant CPSIT_0959 possesses cysteine desulfurase and PLP-binding activity, which removes sulfur from cysteine to produce alanine, and helps chlamydial replication. Our study shows that CPSIT_0959 improve the infectivity of offspring elementary bodies and seems to promote the replication by its product. This phenomenon has inhibited by the PLP-dependent enzymes inhibitor. Moreover, CPSIT_0959 increased expression of Bim and tBid, and decreased the mitochondrial membrane potential of host mitochondria to induce apoptosis in the latecycle for release of offspring. These results demonstrate that CPSIT_0959 has cysteine desulfurase and PLP-binding activity and is likely to contribute to apoptosis of the infected cells via a mitochondria-mediated pathway to improve the infectivity of progeny.     

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N′-cystamine-bis-acrylamide)/pDNA complexes

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)—25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N′-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.     

Molecular mechanisms regulating O-linked N-acetylglucosamine (O-GlcNAc)–processing enzymes

The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion.     

Identification of precursor microRNAs within distal axons of sensory neuron

A set of specific precursor microRNAs (pre‐miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre‐miRNAs are also transported into distal axons to autonomously regulate intra‐axonal protein synthesis. Here, we show that a subset of pre‐miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre‐miRNAs (let 7c‐a and pre‐miRs‐16, 23a, 25, 125b‐1, 433, and 541) showed elevated axonal levels, while others (pre‐miRs‐138‐2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre‐miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

     

Asymmetric inheritance of Cyclin D2 maintains proliferative neural stem/progenitor cells: A critical event in brain development and evolution

Asymmetric cell division and cell cycle regulation are fundamental mechanisms of mammalian brain development and evolution. Cyclin D2, a positive regulator of G1 progression, shows a unique localization within radial glial (RG) cells (i.e., the neural progenitor in the developing neocortex). Cyclin D2 accumulates at the very basal tip of the RG cell (i.e., the basal endfoot) via a unique cis‐regulatory sequence found in the 3′ untranslated region (3′UTR) of its mRNA. During RG division, Cyclin D2 protein is asymmetrically distributed to two daughter cells following mitosis. The daughter cell that inherits Cyclin D2 mRNA maintains its self‐renewal capability, while its sibling undergoes differentiation. A similar localization pattern of Cyclin D2 protein has been observed in the human fetal cortical primordium, suggesting a common mechanism of maintenance of neural progenitors that may be evolutionarily conserved across higher mammals such as primates. Here, we discuss our findings and the Cyclin D2 function in mammalian brain development and evolution.     

Localization of amyloid beta (Aβ1-42) protofibrils in membrane lateral compartments: Effect of cholesterol and 7-Ketocholesterol

Cholesterol plays an important role in the interaction of Alzheimer’s amyloid beta (Aβ) with cell membranes, an important event in Aβ-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aβ with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aβ associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aβ interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aβ-induced toxicity.     

The C-Terminal Domain of SRA1p Has a Fold More Similar to PRP18 than to an RRM and Does Not Directly Bind to the SRA1 RNA STR7 Region

Steroid receptor activator RNA protein (SRA1p) is the translation product of the bi-functional long non-coding RNA steroid receptor activator RNA 1 (SRA1) that is part of the steroid receptor coactivator-1 acetyltransferase complex and is indicated to be an epigenetic regulatory component. Previously, the SRA1p protein was suggested to contain an RNA recognition motif (RRM) domain. We have determined the solution structure of the C-terminal domain of human SRA1p by NMR spectroscopy. Our structure along with sequence comparisons among SRA1p orthologs and against authentic RRM proteins indicates that it is not an RRM domain but rather an all-helical protein with a fold more similar to the PRP18 splicing factor. NMR spectroscopy on the full SRA1p protein suggests that this structure is relevant to the native full-length context. Furthermore, molecular modeling indicates that this fold is well conserved among vertebrates. Amino acid variations in this protein seen across sequenced human genomes, including those in tumor cells, indicate that mutations that disrupt the fold occur vary rarely and highlight that its function is well conserved. SRA1p had previously been suggested to bind to the SRA1 RNA, but NMR spectra of SRA1p in the presence of its 80-nt RNA target suggest otherwise and indicate that this protein must be part of a multi-protein complex in order to recognize its proposed RNA recognition element.     

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.