高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。     

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.     

The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy

Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.     

A novel role for p120 catenin in E-cadherin function

Indirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120-E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.     

A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay.

Human topoisomerase I is composed of four major domains: the highly charged NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.     

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.     

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.