Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.     

Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins

Background

Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region).

Methods

We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions.

Results

LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins—LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins—have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families.

General significance

This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs.     

Human plasma phospholipid transfer protein specific activity is correlated with HDL size: Implications for lipoprotein physiology

To gain further insights into the relationship between plasma phospholipid transfer protein (PLTP) and lipoprotein particles, PLTP mass and phospholipid transfer activity were measured, and their associations with the level and size of lipoprotein particles examined in 39 healthy adult subjects. No bivariate correlation was observed between PLTP activity and mass. PLTP activity was positively associated with cholesterol, triglyceride, apo B and VLDL particle level (r_s = 0.40–0.56, p ≤ 0.01) while PLTP mass was positively associated with HDL-C, large HDL particles, and mean LDL and HDL particle sizes (r_s = 0.44–0.52, p < 0.01). Importantly, plasma PLTP specific activity (SA) was significantly associated with specific lipoprotein classes, positively with VLDL, IDL, and small LDL particles (r_s = 0.42–0.62, p ≤ 0.01) and inversely with large LDL, large HDL, and mean LDL and HDL particle size (r_s = − 0.42 to − 0.70, p ≤ 0.01). After controlling for triglyceride levels, the correlation between PLTP mass or SA and HDL size remained significant. In linear models, HDL size explained 45% of the variability of plasma PLTP SA while triglyceride explained 34% of the PLTP activity. Thus, in healthy adults a significant relationship exists between HDL size and plasma PLTP SA (r_s = − 0.70), implying that HDL particle size may modulate PLTP SA in the vascular compartment.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Vascular endothelial growth factor prevents G93A‐SOD1‐induced motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A‐SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase‐3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1‐induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A‐SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A‐SOD1 toxicity and neuroprotection involves phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

XIAP及survivin 在原发性肝细胞癌组织中的表达及其相关性研究

目的：探讨X 连锁凋亡抑制蛋白（XIAP）和survivin 在原发性肝细胞癌中的表达及两者的相关性。方法：选取本院收治的60 例原发性肝细胞癌患者,应用免疫组织化学染色的方法对肝癌组织及癌旁组织中的XIAP及survivin 的表达进行检测。结果：经 比较,肝癌组织中XAIP 及survivin 的阳性率均显著高于癌旁组织,差异有统计学意义。XIAP 和survivin 的表达强度与肿瘤的大 小无关,但随肿瘤的分化程度的降低而升高,且不同分化程度之间差异有统计学意义;XIAP 和survivin 存在正相关关系。结论： XIAP与Survivin 在肿瘤组织中的高表达在促进肿瘤发生、增殖、转移以及耐药,并且能够降低肿瘤的分化程度,增加肝癌的恶性 程度。此外,两者可能存在协同作用,但两者的相关性及作用机制仍需进一步探讨。     

AEBP1在结直肠癌中的表达及其意义

目的:研究脂肪细胞增强结合蛋白1(AEBP1)在结直肠癌组织中的表达情况,以探索其临床意义。方法:运用免疫组织化学和实时定量PCR技术,对AEBP1在结直肠癌中的表达情况进行检测,并分析其表达量与患者临床病理特征的关系。结果:AEBP1在结直肠正常组织、腺瘤和癌组织中的免疫评分有明显差异(P0.05),而且AEBP1的阳性表达与肿瘤的分期和浸润深度明显相关。基因水平AEBP1的表达在肿瘤组织中明显高于正常组织。结论:AEBP1在结直肠癌组织中存在异常表达,并且可能是结直肠癌早期诊断和浸润深度的肿瘤标志物。