Bioinformatic analysis of protein structure-function relationships: case study of leukocyte elastase (ELA2) missense mutations

Cyclic and congenital neutropenia are caused by mutations in the human neutrophil elastase (HNE) gene (ELA2), leading to an immunodeficiency characterized by decreased or oscillating levels of neutrophils in the blood. The HNE mutations presumably cause loss of enzyme activity, consequently leading to compromised immune system function. To understand the structural basis for the disease, we implemented methods from bioinformatics to analyze all the known HNE missense mutations at both the sequence and structural level. Our results demonstrate that the 32 different mutations have diverse effects on HNE structure and function, affecting structural disorder and aggregation tendencies, stability maintaining contacts, and electrostatic properties. A large proportion of the mutations are located at conserved amino acids, which are usually essential in determining protein structure and function. The majority of the disease-causing HNE missense mutations lead to major structural changes and loss of stability in the protein. A few mutations also affect functional residues, leading into decreased catalytic activity or altered ligand binding. Our analysis reveals the putative effects of all known missense mutations in HNE, thus allowing the structural basis of cyclic and congenital neutropenia to be elucidated. We have employed and analyzed a set of some 30 different methods for predicting the effects of amino acid substitutions. We present results and experience from the analysis of the applicability of these methods in the analysis of numerous genes, proteins, and diseases to reveal protein structure-function relationships and disease genotype-phenotype correlations.     

Controlling fibrous capsule formation through long-term down-regulation of collagen type I (COL1A1) expression by nanofiber-mediated siRNA gene silencing

The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA–poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or the cell penetrating peptides CADY or MPG within the nanofibers (550–650 nm in diameter), a sustained release of siRNA was obtained for at least 28 days (loading efficiency ～60–67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2–3 times as compared to conventional bolus delivery of siRNA (14 days vs. 5–7 days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p < 0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.     

Altered gene silencing and human diseases

Epigenetic regulation of gene expression is mediated through several mechanisms, including modifications in DNA methylation, covalent modifications of core nucleosomal histones, rearrangement of histones and RNA interference. It is now clear that deregulation of epigenetic mechanisms cooperates with genetic alterations in the development and progression of several Mendelian disorders. Here, we summarize the recent findings that highlight how certain inherited diseases, such as Rett syndrome, Immunodeficiency-centromeric instability-facial anomalies syndrome, and facioscapulohumeral muscular dystrophy, result from altered gene silencing.     

Mutations of the human E-cadherin (CDH1) gene

The cell–cell adhesion molecule E-cadherin is well known to act as a strong invasion suppressor in experimental tumor cell systems. Frequent inactivating mutations have been identified for the E-cadherin gene (CDH1) in diffuse gastric cancers and lobular breast cancers. To date, 69 somatic mutations have been reported comprising, in addition to few missense mutations, mainly splice site mutations and truncation mutations caused by insertions, deletions, and nonsense mutations. Interestingly, there is a major difference in mutation type between diffuse gastric and infiltrative lobular breast cancers. In diffuse gastric tumors, the predominant defects are exon skippings, which cause in-frame deletions. By contrast, most mutations found in infiltrating lobular breast cancers are out-of-frame mutations, which are predicted to yield secreted truncated E-cadherin fragments. In most cases, these mutations do occur in combination with loss of heterozygosity (LOH) of the wild-type allele. Inactivating germline mutations of E-cadherin were recently reported for families with early-onset diffuse gastric cancer. Also, at the early stages of sporadic lobular breast and diffuse gastric cancers, E-cadherin mutations were detected, suggesting loss of growth control by such mutations and defining E-cadherin as a true tumor suppressor for these particular tumor types. Hum Mutat 12:226–237, 1998. © 1998 Wiley-Liss, Inc.     

A Bsr BI polymorphism in exon 1C of the human interleukin-1 receptor type I (IL-1RI) gene

We have identified a single nucleotide polymorphism in the 5' region of the human interleukin-1 receptor type I (IL-1RI) gene, a C-->A transversion at position 52 in exon 1C (GenBank accession number AF172151) which creates a Bsr BI restriction endonuclease site. Allele frequencies in a Caucasian population were 0.72 (C allele) and 0.28 (A allele).     

ATP1A1基因沉默抑制人胶质瘤细胞U251侵袭

目的探讨沉默Na+/K+ATP酶A1亚基(ATP1A1)对人U251胶质瘤细胞系侵袭能力的影响及其机制。方法用shRNA-ATP1A1慢病毒感染人U251胶质瘤细胞,RT-q PCR及Western blot分别检测ATP1A1 mRNA和蛋白的表达;MTT法检测细胞体外的增殖;细胞划痕实验及Transwell小室检测细胞的迁移及侵袭能力;Western blot检测基质金属蛋白酶2/9(MMP-2/9)的表达。结果沉默ATP1A1细胞的ATP1A1 mRNA和蛋白表达均受到了明显抑制;细胞的增殖和迁移、侵袭能力也显著受抑(P0.05);MMP-2和MMP-9的表达也明显降低(P0.05)。结论靶向ATP1A1干扰能够明显抑制胶质瘤U251细胞的体外增殖、迁移及侵袭,其机制可能与MMP-2、MMP-9的下调相关,ATP1A1可能作为胶质瘤治疗的一个潜在靶点。     

Haplotype analysis of the human collectin placenta 1 (hCL-P1) gene

Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alpha-helical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5'-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5'-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium ( r(2) > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility.     

Wip1基因沉默对结肠癌细胞化疗敏感性的影响

目的:观察靶向Wip1基因的特异性siRNA序列对结肠癌细胞Wip1基因的抑制效应,探讨Wip1基因沉默对结肠癌细胞化疗敏感性的影响。方法:将Wip1-811 siRNA转染入Wip1高表达的RKO结肠癌细胞株,采用real-time PCR法检测Wip1 mRNA的表达,采用Western blotting方法检测Wip1蛋白表达,采用MTS方法检测结肠癌细胞活力,流式细胞术检测细胞凋亡及细胞周期。结果:Wip1-811 siRNA明显抑制Wip1的表达。抑制Wip1基因表达后,转染组RKO结肠癌细胞对抗肿瘤药物的敏感性增加,5-氟尿嘧啶处理组RKO结肠癌细胞活力由(89.4±6.6)%降至(74.7±3.9)%(P0.05),奥沙利铂处理组细胞活力由(77.9±2.4)%降至(66.7±2.9)%(P0.05)。转染Wip1-811 siRNA后,5-氟尿嘧啶处理组细胞凋亡比例由(7.7±0.5)%升至(12.3±3.2)%(P0.05);奥沙利铂处理组细胞凋亡比例由(14.7±2.1)%升至(34.0±2.1)%(P0.05)。结论:沉默Wip1基因表达能增强结肠癌细胞的化疗敏感性。     

Structural organization of the human carbamyl phosphate synthetase I gene (CPS1) and identification of two novel genetic lesions

Carbamyl Phosphate Synthetase I deficiency (CPSID) is a rare autosomal recessive urea cycle disorder usually characterized by potentially lethal neonatal hyperammonemia. The large (5215 bp) CPS1-cDNA, expressed only in liver and epithelial cells of intestinal mucosa, has been cloned. Until now the CPS1 genomic organization was unknown. Taking advantage of the phylogenetic lineage between the CPS1 gene of Homo sapiens and Rattus norvegicus, we determined the intron-exon organization of the human CPS1 gene. Starting from the ATG codon, the CPS I gene is organized in 38 exons spanning from 50bp to 200 bp. We also report the molecular studies on an Italian patient affected by neonatal CPSD. Two novel genetic lesions (c.1370T>G and c.2429A>G) that lead to the novel amino acid substitutions V457G and Q810R, and the known N1406T polymorphism, were detected in the patient's CPS1 RNA and in genomic DNA isolated from peripheral blood lymphocytes. The characterization of the CPS1 genomic organization will allow the identification of the genetic lesions of CPSD patients, the detection of carriers, better genetic counseling and a more certain, less invasive method of prenatal diagnosis.     

ABCE1基因沉默对人宫颈癌XB1702细胞生物学特性的影响

背景：随着基因工程以及肿瘤生物分子学等新兴学科的发展壮大,基因治疗肿瘤成为一种新的治疗模式。 目的：探讨ABCE1基因沉默对人宫颈癌XB1702细胞的生长、增殖及迁移等方面的影响。 方法：设计及合成ABCE1的siRNA序列,LipofectamineTM 2000转染XB1702细胞。以转染NC siRNA载体细胞为对照组,未转染的宫颈癌XB1702细胞为空白组。通过RT-PCR、Western blot检测RNA干扰后ABCE1 mRNA和蛋白的表达,流式细胞仪检测细胞周期,并通过CCK-8增殖实验、划痕愈合实验、细胞侵袭实验评价沉默ABCE1基因对人宫颈癌XB1702细胞增殖、迁移以及侵袭能力的影响。 结果与结论：实验组ABCE1 mRNA和蛋白表达明显低于对照组和空白组(P < 0.05)。实验组细胞的生长速度明显减慢,细胞被阻滞在G0/G1期,S期细胞数减少。与对照组和空白组相比,实验组XB1702细胞的增殖受到明显抑制、迁移能力和侵袭能力显著下降(P < 0.05)。结果表明特异性干扰ABCE1基因表达可抑制人宫颈癌XB1702细胞的迁移能力,并抑制肿瘤细胞增殖,因此,ABCE1的siRNA序列可能成为治疗宫颈癌的有效靶点。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程