Abstract

P. falciparum possesses the most AT-rich genome sequenced to date, with long AAT repeats coding for asparagines (N) in intragenic regions. As a result, nearly a quarter of its proteome is composed of proteins with poly-N repeats. Since the discovery of its genome, the function of these repeats has remained as one of the biggest mysteries in the biology of this parasite. Furthermore, it has long been known that proteins with N-rich domains aggregate easily, particularly during heat stress (HS). Given that the hallmark of this disease is recurrent febrile episodes, it is not well understood how the parasite survives this stress with an aggregation-prone proteome. Only two of the 1,302 proteins containing poly-N domains in P. falciparum have been experimentally studied. In the first case, Rpn6, a non-enzymatic component of the proteasome, was found to remain stable and active when its 28-residue poly-N domain was removed, during standard and heat stress (HS) conditions, resulting in unaltered parasite growth. This study suggests that poly-N repeats may not play a functional role in the parasite. In the second case, PfHsp110, but not mammalian or yeast homologs, was able to fold the parasite’s putative CDK2-regulatory subunit (possessing an 83 N run) upon heat shock (HS), highlighting the robust specialization that parasite chaperones have developed. Recent discoveries in other organisms whose proteomes are N-rich have shed light on the numerous functions that low-complexity regions, such as poly-N domains, carry out. Mounting data indicate that this is a pervasive phenomenon in the cell, with multiple pivotal roles, including cell recovery, signaling, complex assembly, cellular organization, and transcriptional regulation. Inspired by these discoveries in biology, we decided against the odds to study the role of an imperfect repeat of 98 residues containing 81 N, in the P. falciparum histone acetyltransferase protein, GCN5 (PfGCN5). We observed that the extended low-complexity intrinsically disordered regions in the N-terminus of P. falciparum or P. vivax GCN5 are important for parasite growth. We revealed that the poly-N repeat of PfGCN5 interacts directly with the C-terminal catalytic domain of the protein following cleavage. Deletion of the poly-N repeat in PfGCN5 leads to parasite growth impairment by destabilizing the N-terminal polypeptide after cleavage from the catalytic domain. Interestingly, deletion lines are highly susceptible to stress and display a substantial decrease in histone acetylation. We confirmed PfGCN5 as the enzyme responsible for H3K9ac and newly implicated it in the acetylation of histone variants H3.3 and H4. Moreover, the interactions and localization of PfGCN5 in the context of heat stress are further explored, and the essentiality of two other poly-N repeat-containing proteins is determined. To our knowledge, this is the first study to discover a functional role for a poly-N domain in P. falciparum. Future studies should focus on the role of the poly-N repeat in the SAGA complex and its mechanistic contribution to PfGCN5 function.

Committee Chair

Daniel Goldberg

Committee Members

Alex Holehouse; Daniel Goldberg; David Sibley; Meredith Jackrel; Tamara Doering

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-13-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-4149-4030

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