Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors

Author: Donna Huber

Plasmodium falciparum cGMP-dependent protein kinase interacts with a subunit of the parasite proteasome

ABSTRACT

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, H. M. Kudyba, V. Muralidharan, B. E. Turk, P. Bhanot. 2018. Infection and Immunity. https://doi.org/10.1128/IAI.00523-18

5-Diphosphoinositol Pentakisphosphate (5-IP7) Regulates Phosphate Release from Acidocalcisomes and Yeast Vacuoles

Abstract

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate sodium symporter (TbPho91, and Pho91p, in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation, and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi -loaded giant vacuoles prepared from wild type or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ strains, or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Evgeniy Potapenko, Ciro D Cordeiro, Guozhong Huang, Melissa Storey, Christopher Wittwer, Amit K Dutta, Henning J. Jessen, Vincent J. Starai and Roberto Docampo. 2018. Journal of Biological Chemistry; 293:19101-19112.
doi: 10.1074/jbc.RA118.005884

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation

graphical abstract

Abstract

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure–activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruziLeishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Jennifer L. Woodring, Ranjan Behera, Amrita Sharma, Justin Wiedeman, Gautam Patel, Baljinder Singh, Paul Guyett, Emanuele Amata, Jessey Erath, Norma Roncal, Erica Penn, Susan E. Leed, Ana Rodriguez, Richard J. Sciotti, Kojo Mensa-Wilmot, and Michael P. Pollastri. 2018. ACS Med. Chem. Lett.; 9(10):996-1001. DOI: 10.1021/acsmedchemlett.8b00245

Grad school ready

by Camie Williams

 

Stephen Hajduk with student
PREP@UGA Scholar Jilarie Santos Santiago, center, consults with her faculty mentor Stephen Hajduk, left, and postdoctoral mentor Michael Cipriano. (Photo by Dorothy Kozlowski)

PREP@UGA Scholars program trains next generation of life sciences researchers.

As an undergraduate student in Maryland, Ian Liyayi planned to major in nursing but got lost on the campus tour and found himself in the biochemistry department. He liked that even better. 

When it came to preparing for graduate school, Liyayi didn’t want to get lost along the way, so he applied for the University of Georgia’s competitive PREP@UGA Scholars program to give him more experience before he began applying to doctoral programs.

“This program gets you fully ready for grad school because you get a ton of time in the lab,” said Liyayi, a current scholar who said he enjoyed research experiences in his undergraduate years at Stevenson University but didn’t gain much hands-on laboratory experience on long-term projects. 

“I knew I wanted to do graduate school, but I didn’t feel like I was completely ready,” said Liyayi, a native of Kenya who grew up in Baltimore. “This program seemed like a perfect fit.”

With funding from a National Institutes of Health grant, the PREP@UGA Scholars program was created five years ago. Earlier this year, co-directors Erin Dolan and Mark Tompkins received a $2.1 million, five-year grant renewal, which will continue to fund a cohort each year of six to eight scholars from underrepresented groups or with limited opportunities in the STEM fields at their undergraduate institution. 

To date, 32 students have participated in the program. About one in four later enrolled in a UGA doctoral program, with the remainder going on to graduate programs at other institutions. Programs such as PREP@UGA have helped make UGA the nation’s top public flagship university for the number of doctoral degrees it awards to African Americans.

“Undergraduate students are in the mindset of taking classes, but in grad school they don’t just consume knowledge, they create it,” said Dolan, Georgia Athletic Association Professor of Innovative Science Education in the department of biochemistry and molecular biology, part of the Franklin College of Arts and Sciences. “This program smooths that transition to graduate school and to thinking like a scientist.”

While students spend most of their time in a laboratory, they also go through professional development workshops and benefit from the advice of a faculty mentor and an advanced graduate student or postdoctoral mentor. “It’s a holistic program,” Dolan said. “We focus on the research experience and the career around it.”

For Jilarie Santos Santiago, those mentors have helped her realize her potential in just her first few weeks on campus. “I am stepping out of my comfort zone,” said the graduate of the University of Puerto Rico Humacao, who is currently conducting research in Stephen Hajduk’s lab.

As an undergraduate, Santos Santiago worked on a project for several years to determine a way to thwart parasitic nematodes from destroying the plantain harvest on her home island, but she wanted to explore other areas of life sciences research and learn new techniques before beginning a doctoral program. She also wanted to improve her communication skills, and she’s excited to learn more about the process of publishing research articles.

“The transition to a Ph.D. program, it can be overwhelming. Even this building is confusing when you come from a small college,” Santos Santiago said from her lab in the Davison Life Sciences Complex. “It can be a lot to take in, but if you don’t take the first step, you never do it. This was the right first step for me.”

Tompkins, a professor of infectious diseases in the College of Veterinary Medicine, said his experience as a mentor for a PREP@UGA Scholar last year drove home the impact of the program on students, the research team and on academia as a whole.

“It’s a win-win for the students, the faculty member and the research mentor,” he said. “The perspectives that the scholars bring add a richness to the lab. For the university and academia, the program will have an intangible impact on increasing diversity in the long run.”

Tompkins’ former scholar, Carlie Neiswanger, who has recently begun her doctoral program in pharmacology at the University of Washington, said her experience as a PREP@UGA Scholar was “nothing short of life-changing.” 

After a rocky start to her undergraduate education, she found her passion as a returning student but didn’t believe she had the grades and test scores to make graduate school an option. But her postbaccalaureate experience changed that while providing lessons in independent thinking and problem solving that have given her confidence going into her doctoral program.

“I knew that I wanted to stay in research after graduation, but I was at a loss for what the next steps would be if I wasn’t prepared for graduate school. The PREP program was a near-perfect solution for me,” said Neiswanger, an alumna of Washington State University. “Not only did I get to experience what it means to work full time in a lab while learning to balance things like classes and social life, it truly made me feel prepared for graduate work. … I worked really, really hard, and it paid off. Now I’m ready for the next step.”

 

First published at https://news.uga.edu/grad-school-ready/

Data boost: Using big data to fight disease

Jessica Kissinger

From leisure to health, digital databases can streamline nearly every facet of modern life.

Remember when making travel plans to a single destination took hours? Now booking flights, hotels and rental cars is just a few clicks—and a credit card—away thanks to travel sites like Expedia, Travelocity and others. Travelers get to compare competitors on price, amenities, customer reviews and proximity to popular locations. The sites pull together multiple data points from various sources (such as pricing from the seller, reviews from users, and maps from Google) and organize them for customers to view.

Jessica Kissinger, the director of UGA’s Institute for Bioinformatics and member of the Center for Tropical and Emerging Global Diseases, is doing for infectious disease research what travel sites did for vacation planning.

All over the world, researchers are racing to stop the spread of deadly and debilitating pathogens such as malaria. As those researchers and public health officials determine, or record data about a disease, Kissinger and her colleagues work to make that data accessible and searchable by the global research community for free.

“We take data generated by others and make them better,” says Kissinger, a Distinguished Research Professor of Genetics. More specifically, Kissinger and a team of cell biologists, geneticists and computer scientists pull disease data from a variety of sources, translate them into standard formats and make them searchable.

 

Jessica Kissinger, the director of UGA’s Institute for Bioinformatics, is doing for infectious disease research what travel sites did for vacation planning.

Enabling Discovery

How does building a database fight disease? Data help researchers construct and test their ideas about how to create treatments for diseases or map out ways to halt their spread.

“We don’t give them answers,” Kissinger says. “We give them a framework in which to generate and test hypotheses.”

Kissinger and her team have built databases to take on malaria and other infectious diseases such as toxoplasmosis, cryptosporidiosis and trypanosomiasis. They are also creating tools for studying childhood malnutrition and factors related to disease, and making them accessible to all as they become publicly available. These databases collectively service more than 70,000 unique users a month from more than 100 countries.

To put it simply, her work saves time. It speeds the pace discovery for the next possible solution, the next cure. Without these databases, researchers could spend weeks, months, even years researching existing literature on a disease in the library or recreating work in the lab.

Jessica Kissinger with student

These are tools by biologist for biologists … I think it is that sense of being a member of that community, having your finger on the pulse of what’s going on, that allows you to keep the tools useful. ~ Jessie Kissinger Director, Institute of Bioinformatics

Career Evolution

Kissinger didn’t set out to build databases.

She was trained as a molecular evolutionary biologist, not a computer scientist. “I like to see how molecules change over time,” she says. “When I started in school it was about how a gene or protein evolved.”

It turned out that her field was evolving too. Technology was allowing researchers to understand molecules through bigger data sets. Now, scientists aren’t just looking at individual genes but entire genomes, which are the complete sets of genes in a cell or organism.

As the field evolved, Kissinger learned and embraced the technology. Over time, she shifted her balance away from the so called “wet lab,” where she worked directly with the organisms, to focus mostly on the computer-based “dry lab.”

Her database work started with malaria and continued to expand.

“Now we make 10 different component databases for over 300 organisms (EuPathDB.org), a comparative database to see how conserved genes are across organisms and a new epidemiology database to study the prevalence, spread and factors related to disease in humans (ClinEpiDB.org).” she says.

Kissinger’s team relies on an expert advisory board that helps the researchers customize the databases for each disease community, so they have the largest impact on research. It helps that Kissinger started in a wet lab before diving into informatics.

“These are tools by biologist for biologists,” she says. “We have a lot of computer scientists in the middle, but I think it is that sense of being a member of that community, having your finger on the pulse of what’s going on, that allows you to keep the tools useful.”

 

SUPPORT OUR RESEARCH

Give to the Center Tropical & Emerging Global Diseases General Fund

[button size=’large’ style=” text=’Give Now’ icon=” icon_color=” link=’https://ctegd.uga.edu/give/’ target=’_self’ color=” hover_color=” border_color=” hover_border_color=” background_color=’#b80d32′ hover_background_color=” font_style=” font_weight=” text_align=’center’ margin=”]

 

Originally published at https://greatcommitments.uga.edu/story/data-boost/

Rearranged T Cell Receptor Sequences in the Germline Genome of Channel Catfish Are Preferentially Expressed in Response to Infection

Rearranged V(D)J genes coding for T cell receptor α and β chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vβ2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vβ2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vβ2 PCR products confirmed that these expressed public Vβ2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vβ gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vβ2 and Vβ4 genes retain the intron between the leader and Vβ sequence. This suggests that the germline rearranged TCR Vβ genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline.

Robert Craig Findly, Frank D. Niagro, Ryan P. Sweeney, Alvin C. Camus and Harry W. Dickerson. 2018. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.02117