Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when re-purposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerisation of the RING domain, to switch on the ubiquitination activity of TRIM21 and induce virus neutralisation or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyQ tracts, and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.

SUMMARYTrim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

Mechanical shunting of cerebrospinal fluid (CSF) is an effective treatment for hydrocephalus but is not exempt from complications. A 67-year-old male with a history of normal pressure hydrocephalus (NPH) and ventriculoperitoneal shunting (VPS) one year ago presented with gait disturbance and memory impairment. His head computed tomography (CT) was normal, and the shunting pressure was reduced from 110 to 70 mmH 2 0 with gait and memory improvement. One week later, he reported persistent pressure headaches, which worsen when lying down, accompanied by nausea and vomiting. His neurological examination was notable for a short-stepped wide-based gait. Two generalized seizures were observed. CT cerebral venography revealed sinus venous thrombosis (SVT). After two days, a new CT was performed, and bilateral subdural hygromas were found. The shunting pressure was readjusted to 110 mmH 2 0, and symptom improvement was noted. One week later, CT showed enlargement and bleeding of subdural collections. The drainage system was closed, and the patient continue to recover.The temporal association between pressure adjustment and symptom onset and the evidence of progressive subdural effusions suggest that the decrease of CSF volume by overdrainage led to an increase in cerebral blood volume and the dilatation of the venous sinus, which precipitated thrombus formation.

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