MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Evaluation of the antiparasitic activity of the chitosan-silver nanocomposites in the treatment of experimentally infested pigeons with Pseudolynchia canariensis

This study aimed to evaluate the efficacy of chitosan-silver nanocomposites in the treatment of experimentally infested pigeons with Pseudolynchia canariensis (P. canariensis) with evaluation of different immunological parameters before and after treatment. Therefore, fourteen birds were divided into 2 groups; group1(infested group including 12 birds) which subdivided into 6 sub-groups experimentally infested pigeons 2 pigeons each, and five group of them were treated with chitosan-silver nanocomposites and sub-group number 6 was treated with deltamethrin while, group 2 including two pigeons were kept as control negative ones. P. canariensis flies distributed under the wing and /or under the tail in infested group and these pigeons showed significantly lower RBCs and higher WBCs than that in non-infested pigeons. The cell mediated immune response against experimentally infested pigeons with P. canariensis was studied. P. canariensis infestation in pigeons have a negative impact on pigeon’s blood parameters, increase TNF-α and IL-1β cytokines levels. This study cleared out the role of P. canariensis in the induction of a case of oxidative stress indicated by high level of nitric oxide and malondialdehyde (MDA) with low antioxidant capacity in shape of reduced zinc concentration in the sera of experimentally infested pigeon. Chitosan-silver nanocomposite has a promising effect in the elimination of P. canariensis infestation in pigeons.     

Wheat-yellow pumpkin composite flour: Physico-functional,rheological, antioxidant potential and quality properties of pan and flat bread

The impact of yellow pumpkin powder (YP) substitution (5, 10, and 15%) on wheat flour's physico-functional, pasting, gel texture, and dough rheology was studied. Moreover, the nutritional, organoleptic properties and bioactivity of the composite pan and pita bread were evaluated. An improved water holding capacity was noticed for the blended flour than for control. The pasting parameters were declined significantly (p < 0.05) with increasing YP. Composite flours presented a softer gel texture in the presence of YP. Reduced water absorption, increased dough development time, and lower stability for combined flour dough. The bread with YP depicted increased protein, fat, fiber, and mineral contents, while a reduced volume and specific volume were noticed for pan bread. YP incorporated 5% and did not compromise pan and pita bread's color and overall acceptability. Additionally, composite bread depicted higher total phenolics with enhanced antioxidant activities at the higher substitution of YP.     

Melatonin relieves diabetic complications and regenerates pancreatic beta cells by the reduction in NF-kB expression in streptozotocin induced diabetic rats

Melatonin, a pleiotropic hormone, has many regulatory effects on the circadian and seasonal rhythms, sleep and body immune system. It is used in the treatment of blind circadian rhythm sleep disorders, delayed sleep phase and insomnia. It is a potent antioxidant, anti-inflammatory, free radical scavenger, helpful in fighting infectious disease and cancer treatment. Decreased level of circulating melatonin was associated with an increased blood glucose level, losing the anti-oxidant protection and anti-inflammatory responses. We aimed to evaluate the effect of melatonin administration, in streptozotocin (STZ) induced diabetic rats, on blood glucose level and pancreatic beta (β) cells. Diabetes mellitus was induced in Sprague dawley male rats by the intravenous (i.v) injection of 65 mg/kg of STZ. Diabetic rats received melatonin at a dose of 10 mg/kg daily for 8 weeks by oral routes. The results showed, after 8 weeks of melatonin administration, a reduction in: 1- fasting blood glucose (FBG) and fructosamine (FTA) levels, 2- kidney and liver function parameters, 3- levels of serum triglycerides, cholesterol and LDL-C, 4- malondialdehyde (MDA), 5- NF-κB expression in treated group, 6- pro-inflammatory cytokines (IL-1β and IL-12) and immunoglobulins (IgA, IgE and IgG). Furthermore, an elevation in insulin secretion was noticed in melatonin treated group that indicated β cells regeneration. Therefore, melatonin administration, in STZ induced diabetic rats; reduced hyperglycemia, hyperlipidemia and oxidative stress. Melatonin acted as an anti-inflammatory agent that reduced pro-inflammatory cytokines (IL-1β and IL-12) and oxidative stress biomarkers (MDA). Melatonin succeeded in protecting β cells under severe inflammatory situations, which was apparent by the regeneration of islets of Langerhans in treated diabetic rats. Moreover, these results can open a gate for diabetes management and treatment.     

Regulatory and molecular properties of citrate synthases from methylotrophs

Abstract Gram-negative methylotrophs contain a high- M _r'large' citrate synthase. Gram-positive methylotrophs, on the other hand, contain a 'small' citrate synthase. These differences in M _r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Gram-positive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

表达HIV-1 gag重组鸡痘病毒的构建与筛选

构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。     

沙眼衣原体分泌性蛋白Pgp3滴鼻免疫增强小鼠对衣原体生殖道感染的保护作用

【目的】探讨不同免疫途径沙眼衣原体(Chlamydia trachomatis,Ct)分泌性蛋白Pgp3的免疫保护效果,分析其可能的保护机制,以确定Pgp3蛋白疫苗在Ct疫苗研制中的应用价值。【方法】分泌性蛋白Pgp3经滴鼻或肌注途径免疫雌性Balb/c小鼠,免疫60 d后,阴道接种鼠沙眼衣原体(Chlamydia muridarum,Cm)建立生殖道感染动物模型,在该模型中评价Pgp3蛋白疫苗抗Cm感染的保护效果,并探讨其机制。【结果】滴鼻或肌注免疫后,小鼠血清及生殖道中检测到了特异性抗体;小鼠脾淋巴细胞产生IFN-γ、IL-17及IL-5水平均明显高于对照组,且滴鼻免疫组IFN-γ水平升高较肌注组更显著(P<0.05);Pgp3蛋白滴鼻免疫组小鼠经Cm生殖道感染后,阴道带菌时间明显缩短,输卵管病理改变轻而肌注免疫组其保护作用不明显。【结论】Pgp3蛋白经滴鼻免疫可有效诱导小鼠产生明显的抗Cm保护效应。其可能的免疫保护机制与诱导Th1型为主的细胞免疫应答及高效价的特异性抗体有关,提示Pgp3蛋白疫苗具有潜在的疫苗研究与开发价值。     

GST pull-down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用

为体外验证流感病毒PB1-F2与热休克蛋白Hsp40相互作用,通过两个方向的GST pull-down试验验证PB1-F2与Hsp40的相互作用。构建GST-多肽融合蛋白原核表达载体pGEX-6P-1-PB1-F2和pGEX-6P-1-Hsp40,并在大肠杆菌(E.co-li)BL21中诱导表达;构建真核表达载体pLEGFP-Hsp40及pCAGGS-PB1-F2,并分别转染293T细胞使其表达Hsp40及PB1-F2融合蛋白,然后进行GST pull-down试验验证二者的相互作用。成功地构建了两种蛋白的各种表达载体,经表达、纯化获得了可溶性的GST-多肽融合蛋白,GST pull-down试验正反两方向都证实了PB1-F2与Hsp40的相互作用,初步证实了流感病毒PB1-F2在体外能与Hsp40发生相互作用。