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TRIM21 is an exceptionally versatile ubiquitin ligase that can be directed by antibodies to target oligomeric protein scaffolds, viral capsids, and proteopathic aggregates for intracellular degradation. How the cell degrades these typically resistant substrates remains poorly understood. To address this, we used TRIM21 viral restriction to create a genome-wide phenotypic screen for antibody-dependent capsid degradation. We identify an antimicrobial selective macroautophagy pathway in mammalian cells, which we term "antibody-directed xenophagy" (ADX). We show that this mechanism restricts structurally diverse pathogens, including adenovirus and Salmonella. Using quantitative microscopy, we demonstrate that TRIM21 rapidly intercepts antibody-pathogen complexes, leading to ubiquitin ligase activation. Following this, selective autophagy adaptors are recruited, and viral cargoes are delivered to lysosomes. This process reduces Salmonella pathology and bacterial tissue invasion in mice. We propose that TRIM21 evolved through competition with pathogens to induce autophagy of diverse and complex substrates, potentially explaining its versatility for targeted protein degradation.
We identify an antimicrobial selective macroautophagy pathway in mammalian cells, which we term “antibody-directed xenophagy” (ADX).
genetic, pharmacological, imaging, and degradation evidence converges on selective autophagy in the tested adenovirus system
We show that this mechanism restricts structurally diverse pathogens, including adenovirus and Salmonella.
restriction is shown in two distinct pathogens, but the wording implies broader pathogen-class generality than was tested
TRIM21 ubiquitin ligase activity is required for the incarceration of viral cargoes in autophagosomes and their delivery to lysosomes for degradation.
RING-mutant, ubiquitination, adaptor, LC3, lysosomal, and degradation assays support the mechanism despite a trending direct acidification probe
TRIM21 and RNF213 antibacterial activities are complementary, with the strongest block to proliferation observed in cells that express both ligases and are treated with antibodies.
comparative knockout-cell growth assays support complementary activity, though evidence is confined to the tested Salmonella cell model
Derived from the full evaluation — not a separate score.
Strengths
The genome-wide screen is followed by stable depletion, transient knockdown, complementation, inhibitor timing, adaptor reconstitution, microscopy, and viral-protein degradation assays. These approaches converge on a TRIM21-dependent selective-autophagy mechanism.
↳ Results, Figures 1–3; STAR Methods, Cell library and genome-wide screen
Time-of-addition experiments, proteasome-activity controls, autophagy perturbations, and lysosomal stabilization shift the explanation of antibody-dependent capsid destruction from direct proteasomal degradation toward selective macroautophagy.
↳ Results, Figures 2C–2D and S3F–S3K; Figure S6E–F
The Discussion distinguishes ADX from other antiviral xenophagy mechanisms and experimentally compares TRIM21 with RNF213 in Salmonella restriction. The stated UBE2N uncertainty is also carried into the limitations.
↳ Results, Figure 4E; Discussion, paragraphs 1–2; Limitations of the Study
Limitations
The Key Resources Table lists the catalytic mutant as I18R/M73E, whereas the Results and cloning and complementation methods identify I18R/M72E. The broader evidence base remains convergent, but the central negative-control construct should be reconciled.
↳ Key Resources Table; Results, Figure 2; STAR Methods, Plasmids and cloning and Genetic complementation
The passive-transfer experiment demonstrates TRIM21-dependent serum protection but does not perturb autophagy in vivo. Accordingly, the Abstract's attribution of reduced pathology to “this process” is stronger than the design supports.
↳ Abstract; Results, Figure 4F–G; STAR Methods, Passive transfer experiment
Experimenters were explicitly unblinded, random allocation is not reported for the mouse study, and some microscopy methods do not clearly establish the independent biological unit. Super-resolution colocalization was counted by hand across at least six fields.
↳ STAR Methods, Confocal Microscopy; Structured Illumination Microscopy; Quantification and Statistical Analysis
The central experiments cover adenovirus and Salmonella, with mechanistic depth concentrated in adenovirus. Claims of pan-specificity and broad substrate versatility therefore remain hypotheses for wider testing.
↳ Introduction, final paragraph; Results, Figures 1–4; Discussion, paragraphs 2–3
The core ADX mechanism is supported by a replicated genome-wide screen and multiple orthogonal perturbation, imaging, and degradation assays in Figures 1–3. The score remains below the top band because unblinded assessment, uncertain independent units in some microscopy analyses, and a small passive-transfer experiment introduce identifiable execution limits. Figure 4 supports TRIM21-dependent antibody protection against Salmonella, but it does not directly demonstrate autophagy dependence in mice. Broad claims concerning pan-specificity and targeted-degradation versatility consequently carry less certainty than the adenovirus mechanism itself.
Nabu’s assessment, alongside the field’s view.
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Sound3.7
Confidence mediumThe study meaningfully revises the prior proteasome model by identifying an autophagy-based route supported through screening, perturbation, imaging, and biochemical evidence. Its claims of pan-specificity and broad targeted-degradation relevance extend beyond the two tested pathogens.
“We identify an antimicrobial selective macroautophagy pathway in mammalian cells, which we term ‘antibody-directed xenophagy’.”
The design combines a replicated genome-wide screen with knockout, rescue, pharmacological, imaging, biochemical, and mouse experiments. Explicit non-blinding, hand-counted super-resolution outcomes, uncertain independent units for some microscopy analyses, and a small mouse experiment limit the score.
“Throughout the study, experimenters were not blinded to subject assignments.”
The progression from screen to mechanism and pathogen extension is readily followable. The Abstract and Discussion nevertheless attribute mouse protection to ADX without testing autophagy in vivo and generalize from two pathogen systems.
“This process reduces Salmonella pathology and bacterial tissue invasion in mice.”
The paper engages prior proteasome, xenophagy, RNF213, and targeted-degradation work and states uncertainties concerning screen dropout, UBE2N, and pathogen evasion. It does not integrate the absence of in vivo autophagy testing or the limited pathogen sample into its limitations.
“It is unclear what role UBE2N is playing in the initiation of ADX.”
Caveats4 of 4 checks
The principal results are coherent, but several reporting inconsistencies require correction. Most notably, the catalytic TRIM21 mutant has conflicting residue designations, although the Results and cloning methods consistently identify M72E.
Animal approval, data availability, code availability, and mycoplasma testing are reported. The microscopy methods and Data Availability Statement name different repositories without explaining their relationship.
Flags: 3 declared / 5 total
109 references in manuscript 108 of 108 checkable references found in an index 1 have no canonical index record — counted, but not index-checkable 2 references confirmed by manual review
No retraction notice found in Retraction Watch.
Sources: Retraction Watch ✓
Where this paper’s evidence sits on the path from initial observation to real-world use.
The pathway is characterized in cell systems and supported by one passive-serum mouse experiment, but no therapeutic construct or deployable intervention is tested. Readiness therefore remains early.
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