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Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive ( i.e . non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging. PROTAC development has surged in popularity, however our ability to characterize PROTAC specificity in living cells has lagged behind. Here, the authors develop ProtacID, a flexible proximity-dependent biotinylation (BioID)-based approach to identify PROTAC-protein interactions in living cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD. Nie et al. describe a mechanism underlying the degradation of the histone methyltransferase NSD2 through the recruitment of FBXO22 E3 ligase, providing a chemical probe for NSD2 function study and targeted protein degradation.

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.

Background Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that promotes many cellular processes that lead to cell transformation and tumour development. We previously documented an interaction between Ran-Binding Protein M (RanBPM) and HDAC6 and found that RanBPM expression inhibits HDAC6 activity. RanBPM is part of a putative E3 ubiquitin ligase complex, termed the C-terminal to LisH (CTLH) complex. Here, we investigated the involvement of the CTLH complex on HDAC6 inhibition and assessed the outcome of this regulation on the cellular motility induced by HDAC6. Methods Cell lines (Hela, HEK293 and immortalized mouse embryonic fibroblasts) stably or transiently downregulated for several components of the CTLH complex were employed for the assays used in this study. Interactions of HDAC6, RanBPM and muskelin were assessed by co-immunoprecipitations. Quantifications of western blot analyses were employed to evaluate acetylated α-tubulin levels. Confocal microscopy analyses were used to determine microtubule association of HDAC6 and CTLH complex members. Cell migration was evaluated using wound healing assays. Results We demonstrate that RanBPM-mediated inhibition of HDAC6 is dependent on its association with HDAC6. We show that, while HDAC6 does not require RanBPM to associate with microtubules, RanBPM association with microtubules requires HDAC6. Additionally, we show that Twa1 (Two-hybrid-associated protein 1 with RanBPM) and MAEA (Macrophage Erythroblast Attacher), two CTLH complex members, also associate with α-tubulin and that muskelin, another component of the CTLH complex, is able to associate with HDAC6. Downregulation of CTLH complex members muskelin and Rmnd5A (Required for meiotic nuclear division homolog A) resulted in decreased acetylation of HDAC6 substrate α-tubulin. Finally, we demonstrate that the increased cell migration resulting from downregulation of RanBPM is due to the relief in inhibition of HDAC6 α-tubulin deacetylase activity. Conclusions Our work shows that RanBPM, together with the CTLH complex, associates with HDAC6 and restricts cell migration through inhibition of HDAC6 activity. This study uncovers a novel function for the CTLH complex and suggests that it could have a tumour suppressive role in restricting HDAC6 oncogenic properties.

The primary objective of this study is to investigate whether a novel prosthetic foot with a polycentric ankle, which offers side-to-side adaptability, improves mobility and daily performance in individuals with (1) unilateral above-knee amputation, (2) unilateral below-knee amputation with a lower mobility level (K2) , or (3) bilateral amputation, any level of amputation. Traditional prostheses are primarily designed for forward walking and lack adaptability for navigating uneven terrain, turning, and executing other multidirectional tasks, often resulting in pain, instability, and limited function. The objective of this testing protocol is to better understand the functional impact of a novel foot design that offers more inversion/eversion rotation of the polycentric ankle. Although the foot has the potential to address mobility challenges, it is unknown whether people with different amputations and mobility levels respond differently. The study includes community-based mobility assessments, standardized questionnaires, and participant feedback. Although side-to-side adaptability prosthetic feet have shown benefits for highly active users, this study aims to evaluate the impact on individuals with more significant mobility limitations. Results may guide prosthetic prescription, expand access to advanced prosthetic technology, and inform reimbursement policies, particularly benefiting veterans and service members with combat-related amputations. The study began recruiting in October 2023; as of September 2025, there were 66 participants (17 below the knee, 34 above the knee, and 15 bilateral) who completed the protocol. The clinical trial is expected to be completed by March 2027. Dissemination of results is expected to be completed by December 2027. This procedural study presents a model for evaluating prosthetic feet and other mobility-assistive technologies in real-world contexts, supporting broader efforts to match components to user needs through clinical trials that are grounded in both functional performance and user experience.

A lack of disease-specific knowledge in heart failure (HF) patients is associated with poor adherence to complex medication regimens and non-pharmacological recommendations, especially among individuals with low health literacy. Existing educational tools have been shown to be inadequate, highlighting the need for more accessible and effective alternatives. To develop an educational tool that improves HF knowledge in a more accessible and effective way. An image-based educational tool was created by healthcare professionals and HF patients, drawing on the Health Belief Model (HBM) and utilizing both qualitative and quantitative approaches. Project objectives were defined and content was guided by a literature review and focus group interviews. The tool was refined through iterative optimization using feedback and questionnaires. Thirteen healthcare professionals and eight HF patients participated in the development and refinement of the educational tool. The final tool consisted of seven A3-sized images and accompanying text, addressing the following topics: (1) Cognition of HF; (2) Cause and classification of HF; (3) Inducing factors and prevention of HF; (4)-(5) Medication treatments of HF; (6) Exercise and self-management of HF; (7) \"We are on the same journey\" - a supportive theme for HF patients. The final version of the tool demonstrated high accuracy, readability, usability, and overall satisfaction, with all categories receiving scores of ≥4.5 on a 5-point Likert scale. The image-based educational tool, developed using HBM, effectively meets the needs of HF patients. It shows strong performance in terms of accuracy, readability, usability, and satisfaction, which may ultimately help improve adherence to both pharmacological treatments and non-pharmacological recommendations for HF management.

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental biology field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin β-propeller proteins. Here, we find that, in HeLa cells, CTLH complex E3 ligase activity is dictated by an interplay between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity through the differential association of its β-propeller oligomeric subunits WDR26 and muskelin. WDR26 and muskelin mediate the formation of two distinct CTLH E3 ligase complexes that have different targets. Inhibition of mTOR signalling regulates muskelin-specific CTLH complex activity in human cells and leads to muskelin autoregulation.

The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.