NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438’s molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.
African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the “life cycle” of this critical virulence factor.
Paige Garrison, Umaer Khan, Michael Cipriano, Peter J Bush, Jacquelyn McDonald, Aakash Sur, Peter J Myler, Terry K Smith, Stephen L Hajduk, James D Bangs. mBio. 2021 Jul 27;e0172521. doi: 10.1128/mBio.01725-21.
Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.
Inna Aphasizheva, Juan Alfonzo, Jason Carnes, Igor Cestari, Jorge Cruz-Reyes, H. Ulrich Göringer, Stephen Hajduk, Julius Lukeš, Susan Madison-Antenucci, Dmitri A. Maslov, Suzanne M. McDermott, Torsten Ochsenreiter, Laurie K. Read, Reza Salavati, Achim Schnaufer, André Schneider, Larry Simpson, Kenneth Stuart, Vyacheslav Yurchenko, Z. Hong Zhou, Alena Zíková, Liye Zhang, Sara Zimmer, Ruslan Aphasizhev. Trends Parasitol. 2020 Apr;36(4):337-355. doi: 10.1016/j.pt.2020.01.006. Epub 2020 Feb 28.
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/