Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpn1 (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpn1 are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpn1 in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.     

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.     

尼古丁对卵蛋白致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响

目的 研究尼古丁对卵白蛋白致敏大鼠CD4+T淋巴细胞Th1/Th2平衡的影响.方法 卵清白蛋白(OVA)腹腔注射致敏Wistar大鼠,CD4+T淋巴细胞纯化柱分离大鼠脾脏CD4+T细胞,体外培养,将细胞随机分为4组:对照组、1 μg/ml尼古丁组、10 μg/ml尼古丁组、100μg/ml尼古丁组(各组加等浓度的OVA),不同浓度尼古丁刺激24 h,酶联免疫吸附试验(ELISA)检测细胞培养上清液IFN-γ和IL-4含量,实时荧光定量PCR检测培养细胞T-bet和GATA-3 mRNA的表达.结果 (1)不同尼古丁干预组IFN-γ的表达分别为(113.78±6.06) ng/L、(70.31±7.26) ng/L、( 20.00±2.14)ng/L,均较对照组[(142.30±5.89) ng/L]明显减少,差异有统计学意义(F=265.52,P＜0.01);不同尼古丁干预组IL-4的表达分别为(50.97±3.07) ng/L、(69.49±3.91) ng/L、( 93.63±4.56)ng/L,均较对照组[ (36.91±3.24) ng/L]明显增加,差异有统计学意义(F=128.67,P＜0.01).(2)不同尼古丁干预组T-bet mRNA分别为0.73±0.03、0.57±0.04、0.31 ±0.00,均较对照组(0.98±0.09)明显减少,差异有统计学意义(F=75.76,P＜0.01);不同尼古丁干预组GATA-3 mRNA的表达分别为4.31±0.26、5.16±0.23、1.56±0.14,均较对照组(1.00±0.07)明显增加,差异有统计学意义(F=348.41,P＜0.01).结论 尼古丁可能通过促进转录因子GATA-3 mRNA的表达同时抑制T-bet mRNA的表达,在哮喘Th2过敏性气道炎症中具有一定的作用.     

Streptomyces aureofaciens sporulation-specific sigma factor σ directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, σ^RpoZ, of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P_ren24, P_ren57, and P_ren71, contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P_ren71 was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P_ren71 promoter is recognized by an RNA polymerase holoenzyme containing σ^RpoZ in S. aureofaciens. Sequence analysis of the region directed by P_ren71 revealed a gene, ren71, encoding a protein of 358 amino acids with an M_r 37 770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.