Russell, D. G.; Simwela, N.; Johnston, L.; Sassetti, C.

The GID/CTLH E3 ligase complex is implicated in several biological processes, yet its full substrate repertoire remains poorly defined. We recently identified the complex as a broad modulator of macrophage responses to Mycobacterium tuberculosis (Mtb) infection. Here, we use label-free proteomics and diGly capture analysis of Mtb-infected macrophages to define the GID/CTLH-dependent ubiquitylome. We identify thousands of dynamically altered ubiquitylation sites, with strong enrichment among proteins involved in cellular metabolism and innate immune signaling. Concurrent proteome analysis revealed extensive rewiring in GID/CTLH-deficient macrophages, with >90% of enriched pathways among increased proteins consisting of metabolic targets. Notably, inhibitory phosphatases (PTEN, INPP5D) also emerged as candidate substrates. Functional studies revealed proteasome-dependent stabilization of PTEN and INPP5D in GID/CTLH-deficient macrophages with each phosphatase individually exerting an influence on Mtb intracellular survival. Together, our study defines a GID/CTLH-dependent ubiquitylome in macrophages and identifies the complex as a central regulator of metabolism and antimicrobial immunity.