What is the clinical evidence from this study?

Study design: null. Population: prokaryotic anti-phage defense systems. Intervention: Upx vs. empty vector, Upx inactivating mutant E1176A/K1178A. Primary outcome: Resistance to phage ФV-1 infection (null, 95% CI null, p=null).

What question did this study set out to answer?

This research aims to understand the structural and functional properties of the Upx protein as an anti-phage system.

March 12, 2026Open Access

Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system

Key Result

Upx expression conferred significant resistance to phage ФV-1 infection in E. coli MG1655 but did not provide substantial protection against the other six types of bacteriophages.

Key Points

This research aims to understand the structural and functional properties of the Upx protein as an anti-phage system.
Biochemical analysis of Upx protein
Single-particle electron cryo-microscopy to determine structure
Functional assays to assess nucleic acid binding capabilities
IP-MS experiments to identify interacting phage proteins
Substrate profiling to define physiological preferences
Cryo-EM structure shows Upx contains a C-terminal domain with a PD-(D/E)XK catalytic core.
The N-terminal domain exhibits noncanonical exonuclease activity that is auto-inhibited by the middle domain.
Phage protein gp16 can relieve the MD-mediated inhibition of the NTD, enhancing Upx's activity.

Study Design

Type

null

Structured PICO

Population

E. coli MG1655 and E. coli BL21(DE3) bacterial models, and purified Upx protein

Intervention

Expression of wild-type Upx protein (a PD-(D/E)-XK defense protein)

Comparator

Empty vector control or catalytically inactive Upx mutant (E1176A/K1178A)

Outcome

Phage resistance (assessed via plaque assays) and nuclease activity (assessed via cleavage assays)

Upx is a novel single-protein dual-nuclease anti-phage system that provides direct immunity against specific phages by degrading single-stranded nucleic acid intermediates.

Main Result

Effect estimate: null (95% CI null)

p-value: p=null

Abstract

Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.

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