Deciphering proteolytic processing of carbonic anhydrase 1 from Chlamydomonas reinhardtii

A Chlamydomonas gene CAH1 is responsible for the expression of the secreted protein periplasmic Carbonic Anhydrase 1 (CAH1), which undergoes unique post-translational processing. The pre-protein that has endomembrane targeting sequence and one large and one small subunit separated by a small 35 amino acid stretch called as spacer, undergoes proteolytic processing that cleaves off the targeting sequence and the spacer, giving rise to a mature, glycosylated CAH1 protein. This hetero-tetramer consists of two large and two small subunits attached to each other by disulphide bridges. This research work involves the analysis of the intrinsic factors of the pre-protein that are essential for the proteolytic cleavage of the spacer from the pre-protein. Various potential target sites for the proteases, in and around the spacer region for the proteolytic cleavage were investigated by generating transgenic Arabidopsis plants expressing mutated forms of CAH1 gene. Failure to locate any amino acid sequence/s inside or around the spacer region that controls proteolysis suggests that the proteolysis of the pre-protein is largely independent of the sequence of amino acid residues separating large and small subunits which was supported by successful demonstration of proteolysis of the constructs with the spacer with reverse order of amino acid residues and the spacer with radically different amino acid sequence, in transgenic Arabidopsis plants. We predict possible involvement of more than one protease that act on more than one target sequence in and/or around the spacer region. Analysis of the constructs with progressively reducing number of amino acid residues between the disulfide bond forming cystine residues of large and small subunit suggests that the proteolytic cleavage may be dependent more on the number and the composition of the amino acids between the two cystine residues that form a disulfide bridge between large and small subunits, than the actual sequence. Most interestingly the successful proteolysis of the CAH1 construct lacking previously demonstrated spacer region questions the very requirement of the spacer for the proteolysis of the pre-protein. Even though more research is required to understand this processing better, the post-translational processing undergone by CAH1 may utilize a mechanism that is conserved across the diverse kingdoms making it an interesting and important process to study

An analysis of factors that lead WebCT users to either increase or decrease the use of WebCT GOLD at Iowa State University

This study examined the views and experiences of the instructors and administrators who used WebCT GOLD in the fall 2007 semester. An online survey using multiple-choice and open-ended questions was distributed to a convenience sample of the specified WebCT users at Iowa State University. Descriptive data were collected on demographics, reasons instructors changed their WebCT GOLD usage in the fall 2007 semester as compared to the previous semester, and reasons instructors used certain WebCT GOLD tools the most, while ignoring the rest. The response rate for the survey was almost 18%. The data indicated that a comparatively larger number of respondents increased their use of WebCT GOLD as compared to those who decreased or did not change the WebCT GOLD usage for the fall 2007 semester. Nevertheless, the comments received from the respondents strongly indicate major shortfalls in WebCT GOLD that need to be addressed in order to achieve effective education using WebCT. The statistical data analysis suggest that despite the availability of 20 tools in WebCT GOLD, only the tools such as grade book, announcements, assessments, discussion, and file manager that help reduce work load and increase efficiency were used the most, while instructors ignore the other tools. The findings of this study may help administrators and WebCT GOLD programmers support instructors\u27 effective use of WebCT GOLD, and researchers may learn issues with using WebCT GOLD that need further study

Development of New Breast Cancer Resistance Protein Inhibitors

Chemotherapy is a major form of treatment for cancers. Unfortunately, the majority of cancers are either resistant to chemotherapy or acquire resistance (MDR) during treatment. The most common mechanism by which human cancers develop multidrug resistance is overexpression of certain ATP-binding cassette (ABC) transporters. Breast Cancer Resistance Protein (BCRP / ABCG2), a member of ATP binding cassette family ABCG has been found to confer multidrug resistance in cancer cells. One of the strategies to overcome resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. Several inhibitors of BCRP have been developed in last decade in an attempt to overcome the resistance mediated by this transporter, but there are only very few potent and selective BCRP inhibitors such as Ko143. Aim of the current project was to develop new potent and selective BCRP inhibitors. In this work a total of 131 compounds spanning different chemical classes such as chalcone, flavonoid and quinazoline were investigated. Several biological studies were carried out using these compounds to determine their BCRP inhibitory potential, selectivity, toxicity and efficacy to reverse MDR to certain anticancer compounds. The study of all three classes of compounds revealed structural requirements for BCRP inhibition. In this study, chalcones and benzoflavones showed moderate to good BCRP inhibition with selectivity towards BCRP over other transporters. Amongst chalcones and flavones, 7,8-benzoflavones were the most potent BCRP inhibitors. The structural activity relationship of quinazoline compounds revealed the importance of a 2-phenyl substitution in quinazoline scaffold for BCRP inhibition. The quinazoline compounds were the most active compounds among all classes, some of the quinazolines showing even better activity than Ko143. The results obtained from the study of chalcones, flavonoids and quinazolines as BCRP inhibitors are promising for the ongoing research to find potent BCRP inhibitors

“Cyst-ained” research into Heterodera parasitism

Nematodes are roundworms that constitute the phylum Nematoda. Only a small fraction of nematode genera contains plant-parasitic or animal-parasitic species, while the majority of nematodes are free-living [1]. Heterodera glycines, the soybean cyst nematode, is a plant-parasitic nematode causing major damage to soybean production worldwide. Annual United States yield loss estimates due to H. glycines range up to $1.2 billion, likely making this nematode the most serious pathogen threat to sustainable soybean production [2]. While cyst nematode-resistant soybean cultivars are available, they do not control all H. glycines biotypes present in a given field and, therefore, select for virulent nematode populations that can overcome available resistance genes, leading to a slow but steady erosion of resistance efficacy [3]. Clearly, long-term management of the soybean cyst nematode in modern soybean production will need additional tools, and it is likely that such new tools will be developed from detailed molecular knowledge of the complex Heterodera cyst nematode-plant interactions. This short review provides a snapshot of currently unfolding research discoveries from the genus Heterodera, which also includes other cyst nematodes, particularly the sugar beet cyst nematode H. schachtii, which can infect Arabidopsis and therefore has been used as a model system. Since nematode effectors (the proteins delivered into host plant tissues to mediate parasitism) are at the forefront of nematode–plant interactions, their identification and functional characterization are heavily emphasized in this manuscript

Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1

A virus-induced gene silencing method to study soybean cyst nematode parasitism in Glycine max

Background Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. Findings Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described. Conclusions The VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations

The Cyst Nematode Effector Protein 10A07 Targets and Recruits Host Posttranslational Machinery to Mediate Its Nuclear Trafficking and to Promote Parasitism in Arabidopsis

Plant-parasitic cyst nematodes synthesize and secrete effector proteins that are essential for parasitism. One such protein is the 10A07 effector from the sugar beet cyst nematode, Heterodera schachtii, which is exclusively expressed in the nematode dorsal gland cell during all nematode parasitic stages. Overexpression of H. schachtii 10A07 in Arabidopsis thaliana produced a hypersusceptible phenotype in response to H. schachtii infection along with developmental changes reminiscent of auxin effects. The 10A07 protein physically associates with a plant kinase and the IAA16 transcription factor in the cytoplasm and nucleus, respectively. The interacting plant kinase (IPK) phosphorylates 10A07 at Ser-144 and Ser-231 and mediates its trafficking from the cytoplasm to the nucleus. Translocation to the nucleus is phosphorylation dependent since substitution of Ser-144 and Ser-231 by alanine resulted in exclusive cytoplasmic accumulation of 10A07. IPK and IAA16 are highly upregulated in the nematode-induced syncytium (feeding cells), and deliberate manipulations of their expression significantly alter plant susceptibility to H. schachtii in an additive fashion. An inactive variant of IPK functioned antagonistically to the wild-type IPK and caused a dominant-negative phenotype of reduced plant susceptibility. Thus, exploitation of host processes to the advantage of the parasites is one mechanism by which cyst nematodes promote parasitism of host plants