Biology subjects

Fishburn, A. T.

Publications and source records attributed to Fishburn, A. T..

3 recordsLinked to original sources

bioRxiv2026

Sequence and Structural Alignments Reveal Insights into ANKLE2 Evolution and Function

ANKLE2 is an enigmatic protein with emerging roles in cell division, development, and virus replication. While ANKLE2 orthologs are present in all animals, its domain composition has evolved over time. ANKLE2s two namesake domains, the ankyrin repeat and LEM domains, have clear and defined roles; however nearly all ANKLE2 orthologs have at least three other structured domains with poorly understood purposes. In this study, we performed sequence and structural alignments of ANKLE2 orthologs to improve our understanding of the proteins evolution and function. We identified that ANKLE2s transmembrane domain likely evolved more recently and coincided with loss of VAPA interaction as a membrane anchoring mechanism. We show that despite stark differences in amino acid sequence, the structure of the LEM and ankyrin repeat domains are highly conserved across ANKLE2 orthologs. To investigate ANKLE2s uncharacterized domains, we performed structural alignments to identify similar proteins. This revealed surprising similarities between portions of ANKLE2 and nuclease or nucleic acid-binding proteins. However, ANKLE2 lacks key motifs imparting function in these domains, which was confirmed by experimental interrogation. We further identified that loss of ANKLE2 is correlated with changes in DNA damage response and micronuclei formation. We believe this methodology demonstrates the power of combining structural predictions with classical molecular techniques in exploring poorly understood proteins. ImportanceANKLE2 is a scaffolding protein present in all animals; however much of its function is poorly understood. By evaluating ANKLE2 sequence and structure from many different organisms and comparing its various domains with other proteins, we gain insight into how ANKLE2 evolved and what cellular roles it might be fulfilling. Further, this approach can be used to investigate other understudied or uncharacterized proteins.

molecular biology↗

bioRxiv2025

Yellow Fever Virus Interactomes Reveal Common and Divergent Strategies of Replication and Evolution for Mosquito-borne Flaviviruses

Pathogenic mosquito-borne flaviviruses infect mosquito and human hosts, relying on host protein interactions to replicate, evade immunity, and mediate pathogenesis. Prior proteomic studies mapped such interactions for some flaviviruses, but yellow fever virus (YFV)--a pathogen of resurgent concern--remains understudied. Here, we map YFV interactomes in human and mosquito cells to identify interactions common among divergent flaviviruses or unique to YFV. Functional assays reveal a previously unrecognized YFV restriction factor: RBBP6 inhibits YFV genome replication by interacting with the viral polymerase NS5. We enhance the identification of dual-host interactions using structural modeling and holistic network integration. Extending our holistic approach to other flavivirus interactomes, we distinguish conserved mechanisms of host targeting from those unique to YFV. Contrary to expectations that conserved viral proteins lead to conserved protein interactions, we find that Capsid, a divergent structural protein, shares more host interactions than NS5, a conserved enzyme. Integrating proteomics with complementary analyses defines new principles of host-targeting strategies across flavivirus and host evolution, offering a versatile resource for navigating the complex landscape of flavivirus biology.

systems biology↗

bioRxiv2022

Zika virus NS4A hijacks host ANKLE2 to promote viral replication

Zika virus (ZIKV) is infamous among flaviviruses for its unique association with congenital birth defects, notably microcephaly. We previously mapped ZIKV-host protein interactions and identified the interaction between ZIKV NS4A and host ANKLE2, which itself has established ties to congenital microcephaly. In fruit flies, NS4A induces microcephaly phenotypes in an ANKLE2-dependent manner. This suggests that NS4A interacts with ANKLE2 to dysregulate cell behavior and contributes to abnormal host neurodevelopment. Here, we explore the role of ANKLE2 in ZIKV replication to understand the biological significance of the interaction from the viral perspective. We show that knockdown of ANKLE2 reduces replication of two ZIKV strains, across multiple MOIs and timepoints. We observe that localization of ANKLE2 is drastically shifted to sites of NS4A accumulation during infection. We investigate which domains of ANKLE2 mediate this behavior and the interaction with NS4A. Using co-immunoprecipitation, we show that deletion of either the transmembrane or LEM domain has little impact on the interaction, but deletion of both significantly reduces interaction with NS4A. We show that the C-terminal transmembrane domains of NS4A stabilize the interaction with ANKLE2. Finally, we explore this interaction in other flaviviruses and observe ANKLE2 interacts with NS4A across four additional mosquito-borne flaviviruses. Together, these results suggest NS4A interacts with ANKLE2 through a combination of its transmembrane and LEM domains, bringing it to sites of ZIKV replication to promote replication through an unknown mechanism. Taken together with our previous results, our findings indicate that, in the process of hijacking ANKLE2 for replication, ZIKV disrupts its physiological function to cause disease. ImportanceThe ZIKV epidemic led to the astonishing revelation that congenital ZIKV infection is associated with devastating birth defects, including microcephaly. Microcephaly is the condition in which head and brain size are severely reduced, and is often accompanied by intellectual disability. The molecular mechanisms by which ZIKV replicates and causes microcephaly are still incompletely understood. We previously identified the protein interaction between ZIKV NS4A and host ANKLE2, which is associated with congenital microcephaly. In flies, NS4A induces microcephaly in an ANKLE2-dependent manner, suggesting this interaction is crucial for ZIKV pathogenesis. Here, we explore the relevance of this physical interaction for virus replication. We find that ANKLE2 promotes ZIKV replication, concentrates at sites of NS4A accumulation during infection, and interacts with NS4A via its N-terminal domain. Thus, this represents a rare example of a ZIKV-host protein interaction that impacts both disease and virus replication.

molecular biology↗