Ubiquitylation controls macrophage response to tuberculosis

Macrophages are immune cells that engulf and try to kill Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis. How these cells reprogram their metabolism and immune signals during infection is a major question in TB research. David G. Russell and his team focused on a cellular machine known as the GID/CTLH E3 ligase complex, which tags proteins with ubiquitin to control their stability and activity. Although the complex had been linked to several biological processes, its full set of targets in immune cells was unclear. To fill that gap, the researchers analyzed infected macrophages to map the proteins and specific ubiquitylation sites that depend on GID/CTLH. Their goal was to figure out how ubiquitylation by this complex influences both the metabolic pathways inside macrophages and the innate immune signaling that fights Mtb. The work builds on previous findings that the GID/CTLH complex broadly modulates macrophage responses to Mtb infection and aims to define the biochemical changes that underlie those effects.

To define the GID/CTLH-dependent ubiquitylome, the team used label-free proteomics combined with diGly capture analysis of Mtb-infected macrophages. These methods let them identify and quantify ubiquitylation on thousands of sites across many proteins without the use of labeling reagents. The diGly capture approach specifically isolates peptides bearing the di-glycine remnant that marks ubiquitylation sites, giving a detailed view of dynamic ubiquitin modifications during infection. The data revealed thousands of dynamically altered ubiquitylation sites, with a strong enrichment among proteins involved in cellular metabolism and innate immune signaling. Parallel proteome analysis in GID/CTLH-deficient macrophages showed extensive rewiring: more than 90% of enriched pathways among increased proteins were metabolic targets. Among candidate substrates that stood out were the inhibitory phosphatases PTEN and INPP5D. Follow-up functional studies showed proteasome-dependent stabilization of PTEN and INPP5D in GID/CTLH-deficient macrophages, and each phosphatase individually affected Mtb intracellular survival.

Taken together, these findings position the GID/CTLH complex as a central regulator that links ubiquitylation to both metabolism and antimicrobial immunity in macrophages. By mapping the GID/CTLH-dependent ubiquitylome, the study highlights how protein tagging reshapes metabolic pathways and immune signaling during Mtb infection. The identification of PTEN and INPP5D as candidate substrates is especially notable because changes in their stability were tied to differences in bacterial survival inside macrophages. That connection suggests a mechanism by which ubiquitylation can tune immune cell behavior via control of specific phosphatases and metabolic enzymes. While the work is rooted in molecular analysis of infected cells, it points toward new angles for thinking about host-directed strategies: altering ubiquitylation pathways or the activity of enzymes like PTEN and INPP5D could shift macrophage metabolism and improve control of Mtb. Overall, the study defines a detailed ubiquitylation landscape with clear links to antimicrobial function and metabolism in infected macrophages.