Progress 10/01/12 to 09/30/17
Outputs
Target Audience:The target audience consists of the international malaria research community, in particular those who are interested in a molecular understanding of metabolic pathways in the parasite. Changes/Problems:Due to the unforseen result that one of our enzymes of interest was dispensible and therefore not of potential interest from the point of view of parasite physiology or drug development, investment of effort in Objective 3 was less than initially anticipated. This opened up an opportunity to investigate other types of metabolic enzymes in the parasite. We therefore undertook efforts to identify and characterize parasite members of the serine hydrolase superfamily on a proteomic scale. We have identified several serine hydrolases that are essential for parasite growth and replication. The overall goal remained the same: to understand from a biochemical perspective how the malaria parasite thrives in human red blood cells and to develop new approaches to anti-malarial drug development. What opportunities for training and professional development has the project provided?The following training and professional development activites took place during the project period: 1. Training of an undergraduate student in biochemical research. Mr. Matthew Rosati conducted research in my laboratory from 2012 - 2013 while an undergraduate student at Virginia Tech. Mr. Rosati's training included fundamental biochemical approaches to enzyme characterization, which included recombinant protein expression and purification and the analysis of steady-state enzyme kinetics. Mr. Rosati was trained to use an ultra-high pressure liquid chromatography system, a state-of-the-art instrument for separating and quantifying biomolecules. Mr. Rosati was responsible for the progress in Objective 1 and achieved a high level of accomplishment. His research was recently published with Mr. Rosati as the first author, which speaks to his essential contribution to the execution of the project. 2. Training of a graduate student. Mr. Rubayet Elahi was trained in techniques of protein expression using the baculovirus system while performing a 10-week rotation in my laboratory. Mr. Elahi joined my research group in late 2015. During the past year, his training has continued in the field of chemical biology and biochemistry and he is responsible for the identification and characterization of novel serine hydrolases. 3. Professional development of Ms. Christie Dapper, Laboratory & Research Specialist II, Senior. Ms. Dapper has participated in the identification and characterization of novel serine hydrolases. She has been trained in cutting-edge techniques involving the use of chemical probes and enzyme inhibitors to profile the serine hydrolase enzyme superfamily in the malaria parasite. How have the results been disseminated to communities of interest?Three papers were published, one in the Journal of Biological Chemistry and two in the journal Molecular and Biochemical Parasitology. The publication audience is: i) a broad spectrum of molecular life scientists (Journal of Biological Chemistry); ii) scientists interested in molecular and biochemical processes in protozoan parasites (Molecular and Biochemical Parasitology). Results from the project were presented as posters at the following scientific meetings: 1. The Future of Malaria Research, November 7, 2017, Johns Hopkins Malaria Research Institute, Baltimore, MD. This meeting focusing on the parasite that causes malaria is regional in scope. R. Elahi (graduate student) was the presenter. 2. Experimental Biology Conference, April 20-24, 2013, Boston, MA. This is the annual meeting of the American Society for Biochemistry and Molecular Biology and is national/international in scope. M. Klemba was the presenter. 3. ACC "Meeting of the Minds", April 3-5, 2014, Pittsburgh, PA. This meeting focuses on undergraduate research and is regional in scope. M. Rosati (undergraduate student) was the presenter. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Research during the project period revealed the roles of two amino acids in the malarial aminopeptidase PfA-M1 in sculpting substrate specificity. These findings provided insight into the mechanism of substrate selection by the enzyme, leading to a deeper understanding of its physiological role in the parasite. They also suggested strategies for the design of potent inhibitors as novel anti-malarials. A paper describing this work was published in 2017. We also determined that a distantly-related malarial aminopeptidase, termed PfAPN2, is dispensible during the pathogenic red blood cell replication stage of the parasite. This result indicates that this enzyme would not be a productive target for anti-malarial drug discovery. Given these findings, we reduced our effort on this aspect of the project. We have expanded our enzyme discovery and characterization efforts beyond aminopeptidases to include enzymes of the serine hydrolase superfamily, many of which are expected to have critical roles in the metabolism of parasite macromolecules and thereby serve as potential drug targets. Methods for the identification of novel parasite serine hydrolases through state-of-the-art chemical biology and proteomic approaches have been developed and are being implemented to uncover the roles of previously unknown parasite enzymes. Results for the specific objectives identified in "Major goals of the project" are detailed below. Objective 1: Contributions of two "cap" residues to the S1 subsite specificity of M1-family aminopeptidases. This was the first study to systematically address the role of cap residues in aminopeptidase specificity. The approach taken was to mutate the two cap residues of the malarial aminopeptidase PfA-M1 and to assess the effects of the mutations on the steady-state kinetic behavior of the enzyme in vitro. The results revealed that the cap residues are critical for the binding of peptide substrates carrying certain types of functional groups, and that they enhanced catalytic activity of the enzyme against a broad range of substrates. From an evolutionary perspective, our findings suggested that aminopeptidase specificity can change rapidly in response to selective pressures through naturally-occuring mutations in the amino acids that make up a critical substrate binding pocket in the enzyme. Objective 2: Determine the effects of S1 subsite cap mutations on PfA-M1 function in vivo. The approach taken was to introduce the cap mutations made in Objective 1 into the parasite's single genomic copy of the enzyme. This was not a trivial experiment to undertake in the human malaria parasite Plasmodium falciparum and required many months to effect the desired genomic transformation. While we found that the cap mutants could be introduced individually into the parasite genome, we have not been able to isolate viable parasites carrying both cap mutations together. This result validates the functional importance of the two cap residues for enzyme function in vivo and provides a physiological context for the in vitro results reported in Objective 1. Objective 3: Elucidate the function of PfAPN2, a highly divergent M1-family aminopeptidase with a possible role in the maturation of the blood-stage parasite. Investigation into PfAPN2 was initiated by attempting a genetic knockout of this enzyme in the pathogenic blood stage of the parasite. Surprisingly, we found that PfAPN2 was dispensible with no apparent consequence for parasite growth and replication in culture, which disproved our initial hypothesis for a critical role in the blood stage. These findings indicated that any essential role for PfAPN2 must occur during other stages of the parasite life cycle, such as those in the mosquito host. As a result, PfAPN2 would not be a useful target for anti-malarial chemotherapy in humans. We also attempted to express recombinant PfAPN2 to determine whether it was an authentic aminopeptidase, however these efforts were unsuccessful in that they did not yield properly-folded native enzyme.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Rosati, M, Dalal, S & Klemba M. 2017. Two cap residues in the S1 subsite of a Plasmodium falciparum M1-family aminopeptidase promote broad specificity and enhance catalysis. Molecular and Biochemical Parasitology vol. 217 pp 7-12.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience is the community of scientists who are interested in understanding the biochemistry of the human malaria parasite Plasmodium falciparum during its growth and replication within human red blood cells. Changes/Problems:As noted above, there has been a shift of emphasis in order to pursue a new, exciting avenue of research. The modification involves the type of enzyme studied, namely lipases rather than aminopeptidases. However, the original aims of the project remain highly relevant. Both types of enzymes are catabolic enzymes expressed in the malaria parasite during its parasitism of human red blood cells. Both are involved in important biochemical transformations that take place during parasite development. The larger goal remains as to understand from a biochemical perspective how the malaria parasite thrives in human red blood cells and to develop novel modalities for anti-malarial drug development. What opportunities for training and professional development has the project provided?The project has provided the opportunity for professional development of Ms. Christie Dapper, Laboratory & Research Specialist II, Senior. In executing the described studies, she has learned many new experimental techniques and has increased her knowledge of the biochemistry of the malaria parasite. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will pursue the following objectives: 1) Identification of novel malarial lipases: we will leverage our results over the last reporting period to identify 3-4 putative malarial lipases. This will be accomplished by labeling the lipases with an affinity probe followed by affinity purification and mass spectrometric identification. 2) Recombinant expression and characterization of novel malarial lipases: in order to define the roles of the identified lipases, we will attempt to express them in prokaryotic or eukaryotic expression systems. Recombinant enzymes will be biochemically characterized to establish their substrate specificities and to set the stage for inhibitor development. 3) Functional analysis of novel malarial lipases: to gain insight into the biological functions of these enzymes, we will undertake gene disruption studies to determine whether they are essential for growth in human red blood cells. We will also localize the lipases within the parasitized red blood cell, which will provide important clues into their roles.
Impacts
What was accomplished under these goals?
During our studies with aminopeptidases, the opportunity arose to take the project in a new direction. We have shifted our attention to another class of enzyme termed "serine hydrolases" that, like aminopeptidases, contributes to the catabolism of biomolecules in the malaria parasite. The overarching aim remains the same: namely, to understand biochemical transformations taking place during the growth and replication of the malaria parasite Plasmodium falciparum within human red blood cells. Project progress over the reporting year includes the following: 1) Utilization of a fluorescent chemical probe to rapidly and efficiently profile the serine hydrolase proteome during parasite development. We have identified over 30 distinct serine hydrolases that are expressed in the parasite or exported to the red blood cell, the vast majority of which have not been previously characterized. 2) Functional annotation of the parasite serine hydrolases has been accomplished by assembling a large panel of serine hydrolase inhibitors of diverse specificities and by conducting competition experiments with the chemical probe described above. This effort has led to the identification of a number of putative parasite lipases, which are enzymes that catabolize cellular lipids. These lipases are of interest as potential novel drug targets. 3) Development of a fatty acid uptake and lipid incorporation assay utilizing a fluorescent fatty acid derivative. This functional assay will help us to define the roles of lipases and to determine the effects of the inhibitors described above.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience comprises scientists interested in the biochemistry of the human malaria parasite Plasmodium falciparum during its infection of human erythrocytes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training opportunities for two individuals: 1. Mr. Matthew Rosati is an undergraduate who has been conducting research on a part-time basis in my laboratory since 2012. He has been working on Objective 1 and will be the first author on the manuscript that we are preparing. 2. Mr. Rubayet Elahi was a first-year graduate student who worked on Objective 3 during a 10-week rotation in my laboratory. How have the results been disseminated to communities of interest?Results have been disseminated to the malaria research community through the publication of a manuscript (see Products). What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will focus on Objective 2 and 3. Objective 2: We will undertake a genetic and biochemical analysis of transgenic parasite lines to determine: a) whether the introduction of PfA-M1 cap mutants into the parasite genomic copy of the pfam1 gene is tolerated; and b) if so, what effect the mutations have on the production of amino acids from host cell hemoglobin. Objective 3: We will continue our efforts to develop eukaryotic expression systems that are useful for expressing active, recombinant malarial proteins. We will investigate whether recombinant PfAPN2 can be produced. If successful, we will characterize its catalytic activity and substrate specificity.
Impacts
What was accomplished under these goals?
We have made progress on all three objectives: Objective 1: Biochemical analysis of the roles of "cap" residues in determining substrate specificity in a malarial aminopeptidase called "PfA-M1" has been completed. The data are being prepared for publication. Objective 2: We have progressed in the development of an assay to follow the kinetics of malarial hemoglobin catabolism in vivo. This work was published during the reporting period. Because the aminopeptidase PfA-M1 has a key role in producing amino acids from hemoglobin, this assay will provide a foundation for analyzing the effects of S1 subsite cap mutations in vivo. Objective 3: We have developed proficiency in the use of a baculovirus/insect cell expression system, which holds promise for the generation of the recombinant aminopeptidase PfAPN2. As this aminopeptidase has highly unusual sequence features, determining its biochemical characteristics will be of interest in order to understand how extreme sequence variation has impacted its enzymatic properties.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Dalal, S & Klemba. 2015. M. Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo. Molecular and Biochemical Parasitology, vol 201, pp. 116-22.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Project results were disseminated to the scientific community at an undergraduate research meeting, "ACC Meeting of the Minds", that took place in Pittsburgh, PA on April 3-5, 2014. Mr. Matthew Rosati, an undergraduate student working on the project, presented a poster at this meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? An undergraduate student, Matthew Rosati, continued to work on this project during the reporting period. He gained experience in enzyme structure-function studies. By the time Mr. Rosati graduated from Virginia Tech (May 2014), he had attained a high level of proficiency and competence in this area. How have the results been disseminated to communities of interest? Mr. Rosati presented a poster describing his work on the project at an conference focusing on undergraduate research, "ACC Meeting of the Minds", that was held in Pittsburgh, PA on April 3-5, 2014. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have completed the data collection stage for Objective 1. Mr. Rosati, an undergraduate student, has obtained substrate specificity data for six variants (mutants) of the malarial aminopeptidase. We will be preparing a manuscript for submission to a peer-reviewed journal.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audience was primarily two sectors of the biomedical research community: i) scientists interested in the biochemical workings of the human malaria parasite Plasmodium falciparum and the validation of novel anti-malarial drug targets; and ii) those studying the structure-function relationships of proteolytic enzymes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided a robust training experience for an undergraduate Biochemistry major, Matthew Rosati, who worked on this project for academic credit in Fall 2012, Spring 2013 and Fall 2013 semesters. He was also funded by a Fralin Summer Undergraduate Research Fellowship, which provided a stipend for 10 weeks of full-time effort on the project in the summer of 2013. Mr. Rosati's work focused on Objective 1. He has been trained in all the procedures needed to pursue this objective. He has generated a substantial amount of data and it is anticipated that his studies will be published within the next year. This project also provided a training experience for a first-year Biochemistry graduate student, Feng Li, who spent a 12-week rotation generating and purifying novel PfA-M1 mutants. How have the results been disseminated to communities of interest? These results have been disseminated to the communities of interest (the scientific community) in two ways. First, our initial studies on determinants of specificity in PfA-M1 were published in the Journal of Biological Chemistry (see Products). Second, these results have been presented on a poster at the annual Experimental Biology conference, which draws scientists from around the world. (See Products). In addition, Mr. Rosati has presented his work at a research symposium at Virginia Tech for recipients of the Fralin Summer Research Fellowship award (August 2013). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have made progress on all three of the research objectives during the reporting period. Progress is described for each objective below. Objective 1: Six variants of PfA-M1 were made, each having specific mutations (changes) in one or both of the two S1 subsite cap residues. The effects of these mutations are being characterized by determining the kinetic properties of the mutant enzymes against a panel of dipeptide substrates. While these studies are still in progress, our results so far indicate that each of the two cap residues makes an important contribution to defining the overall specificity of the enzyme. Obective 2: We have attempted to introduce the S1 subsite cap mutations into the chromosomal copy of the PfA-M1 gene in cultured Plasmodium falciparum. Thus far, we have not been able to isolate parasites that have incorporated the desired mutations. This may be because the mutations are interfering with the function of the enzyme in the parasite and therefore parasites carrying these mutations are not viable. Efforts to isolate the desired parasite lines are continuing. Objective 3: We have succeeded in disrupting the gene coding for the cryptic aminopeptidase PfAPN2. This result indicates that this protein is not required during the parasite's replication cycle in red blood cells. At this time we have downgraded our efforts on this objective in order to focus on the essential enzyme and potential drug target PfA-M1 (Objectives 1 and 2).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Dalal, S, Ragheb, RT, Schubot, FD & Klemba, M. A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization. Journal of Biological Chemistry, vol 288, pp 26004-12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Klemba, M, Ragheb, D, Schubot, F & Dalal, S. A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization. Poster, Experimental Biology Conference, April 20-24, 2013, Boston, MA
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