Event Title

Expression of wild-type and mutant constructs for the Plasmodium BEM46-like protein (PBLP).

Research Mentor(s)

Anna Groat-Carmona

Description

Plasmodium infections, commonly referred to as malaria, occur when Anopheline mosquito vectors transmit the obligatory intracellular protists into a mammalian host. A crucial phase within the parasite’s complex life-cycle is the liver-stage (LS). The LS leads to an aggressive parasitic amplification event, which results in the production of thousands of blood-stage parasites that are responsible for causing symptomatic malarial infections, and thus, this stage of the parasite’s life cycle is considered to be a pivotal objective for drug interventions. An understanding of the proteins involved in LS advancement is essential to a greater comprehensive understanding of the morphological alterations that occur on the parasite plasma membrane (PPM) during the mosquito- to LS transition. The Plasmodium BEM46-like protein (PBLP) has previously been shown to be an important determinant for both in vitro and in vivo invasive-stage morphogenesis. However, the enzymatic function of PBLP has not yet been characterized. BEM46 proteins have a conserved amino acid motif in the α/β-hydrolase superfamily, which includes a large collection of enzymes though very few possess a known biological function. Our research suggests that the putative active site of PBLP resembles that of a typical α/β-hydrolase domain, which is consistent with the predicted active site associated with other BEM46 proteins. Currently, we are in the process of employing molecular cloning techniques using an Escherichia coli bacterial protein expression vector to express and purify both wild-type and mutant PBLP constructs. These mutant PBLP constructs have been generated with alterations to the putative active site of the protein using overlap-extension PCR in an attempt to disrupt the catalytic triad that was previously predicted. Our aim is to confirm the putative active site of PBLP using biochemical analyses of the α/β-hydrolase domain, which will allow for comparative analyses of our wild-type protein and the effect that mutations to the catalytic triad may have on protein activity. Other BEM46 proteins have been implicated in signal transduction pathways that result in actin organization at the plasma membrane, which we suspect is why PBLP appears to modulate cell surface morphology in multiple systems. These studies will aid our endeavor to identify the potential downstream targets of PBLP so that we can employ a bioinformatics approach to search for candidate protein interactive partners in the Plasmodium genome.

Document Type

Event

Start Date

May 2018

End Date

May 2018

Location

Biology

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Language

English

Format

application/pdf

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Expression of wild-type and mutant constructs for the Plasmodium BEM46-like protein (PBLP).

Biology

Plasmodium infections, commonly referred to as malaria, occur when Anopheline mosquito vectors transmit the obligatory intracellular protists into a mammalian host. A crucial phase within the parasite’s complex life-cycle is the liver-stage (LS). The LS leads to an aggressive parasitic amplification event, which results in the production of thousands of blood-stage parasites that are responsible for causing symptomatic malarial infections, and thus, this stage of the parasite’s life cycle is considered to be a pivotal objective for drug interventions. An understanding of the proteins involved in LS advancement is essential to a greater comprehensive understanding of the morphological alterations that occur on the parasite plasma membrane (PPM) during the mosquito- to LS transition. The Plasmodium BEM46-like protein (PBLP) has previously been shown to be an important determinant for both in vitro and in vivo invasive-stage morphogenesis. However, the enzymatic function of PBLP has not yet been characterized. BEM46 proteins have a conserved amino acid motif in the α/β-hydrolase superfamily, which includes a large collection of enzymes though very few possess a known biological function. Our research suggests that the putative active site of PBLP resembles that of a typical α/β-hydrolase domain, which is consistent with the predicted active site associated with other BEM46 proteins. Currently, we are in the process of employing molecular cloning techniques using an Escherichia coli bacterial protein expression vector to express and purify both wild-type and mutant PBLP constructs. These mutant PBLP constructs have been generated with alterations to the putative active site of the protein using overlap-extension PCR in an attempt to disrupt the catalytic triad that was previously predicted. Our aim is to confirm the putative active site of PBLP using biochemical analyses of the α/β-hydrolase domain, which will allow for comparative analyses of our wild-type protein and the effect that mutations to the catalytic triad may have on protein activity. Other BEM46 proteins have been implicated in signal transduction pathways that result in actin organization at the plasma membrane, which we suspect is why PBLP appears to modulate cell surface morphology in multiple systems. These studies will aid our endeavor to identify the potential downstream targets of PBLP so that we can employ a bioinformatics approach to search for candidate protein interactive partners in the Plasmodium genome.