MRC Protein Phosphorylation and Ubiquitylation Unit

Selective autophagy is a fundamental protein quality control pathway that safeguards proteostasis by degrading damaged or surplus cellular components, particularly under stress. This process is orchestrated by selective autophagy receptors (SARs) that recruit specific cargo for degradation. Although significant strides have been made in understanding the molecular framework of selective autophagy, the diversity of SAR repertoires across species remains largely unexplored. Through a comparative interactome analysis across five model organisms, we identified a suite of conserved and lineage-specific SAR candidates. Among these, we validated coupling of ubiquitin to endoplasmic reticulum degradation- and protein rich in the amino acids E, L, K, and S-domain-containing SAR (CESAR) as a conserved SAR critical for proteostasis under heat stress. CESAR specifically facilitates the degradation of ubiquitinated protein aggregates and is indispensable for heat stress tolerance. Altogether, our study establishes a robust pipeline and a rich resource for SAR discovery. It also positions CESAR as a pivotal regulator of proteostasis, with broad implications for improving stress resilience in plants.

Genetic variants that hinder post-translational protein modifications by UFM1, UFMylation, cause encephalopathies. UFMylation regulates endoplasmic reticulum (ER) homeostasis, but how UFMylation deficiencies cause selective neurological defects is unknown. Using murine UFM1-deficient neurons, we investigated two types of UFMylation pathologies, UFM1 loss and expression of a pathogenic UFM1-R81C variant. We found that UFM1-deficiency confounds neuron development and synapse function. Mechanistically, UFM1 loss is associated with induction of ER stress, activation of the unfolded protein response (UPR) pathway, and reduced protein translation. These defects are rescued by wild-type UFM1, but only partially by UFM1-R81C. UFM1-deficient and UFM1-R81C-expressing neurons display distinct responses to ER stress, indicating that UFM1-R81C is not merely a loss-of-function variant. Exploring therapeutic options, we show that Trazodone, an inhibitor of the UPR, restores protein translation solely in UFM1-R81C-expressing neurons, and increases synapse numbers in both UFM1-KO and UFM1-R81C-expressing neurons. Our study unveils a pivotal role for UFMylation in neuronal development, provides a molecular understanding of the signaling mechanisms altered in UFM1-associated encephalopathies, and offers important insights into potential treatments for these disorders.

Schematic representation comparing two trajectories of tau pathology. The S305N mutation promotes a 4R isoform shift, cytoskeletal damage and synapse loss, and accumulation of soluble hyperphosphorylated tau. Tau remains soluble even at old ages. In contrast, the P301S mutation generates hypophosphorylated, seed-competent tau that forms fibrils. The effect of amyloid is slow in the S305N, but results in accelerated acceleration of pathology in the P301S. Figure made with Biorender.com .

EML4-ALK is a common oncogenic driver of non-small cell lung cancer. Distinct EML4-ALK variants cause different rates of disease progression, with patients expressing variant 3 (V3) exhibiting accelerated metastasis. Cells expressing EML4-ALK V3 develop a mesenchymal-like morphology and enhanced migration that is dependent on the NEK9 and NEK7 kinases. However, downstream substrates of these kinases relevant to these phenotypes are largely unknown. Here, we show that the actin-binding protein cortactin is phosphorylated by NEK7 within the F-actin-binding region (ABR) and that depletion of cortactin abrogates the morphological and migration phenotypes induced by EML4-ALK V3. Expression of constitutively active mutants of NEK9 or NEK7 causes similar cortactin-dependent morphological and migration changes. Cortactin co-localises with NEK7 and EML4-ALK V3 at branched filopodia-like extensions that are also generated upon expression of a cortactin protein with phospho-mimetic mutations in the ABR. In contrast, phospho-null mutations dissociate cortactin from F-actin. We propose that EML4-ALK V3 alters cell morphology and promotes directed cell migration by modulating the actin cytoskeleton via NEK7-mediated phosphorylation of cortactin within its ABR.

Abstract Congenital vascular malformations are commonly caused by aberrant, genetic activation of class I phosphoinositide 3-kinase (PI3K) signalling. Advances in mechanistic understanding and therapeutic targeting of these disorders will be accelerated by high-fidelity, human disease models. Building on a previously optimised differentiation strategy, we present a validated workflow for PI3K inhibitor-free generation of arterial- and venous-like endothelial cells from human induced pluripotent stem cells (iPSCs) under defined, xeno-free conditions. We further report experimental analyses of endothelial maturation under flow, culture duration-dependent stability, and downstream molecular and phenotypic characterisation. By providing a reproducible human system for mechanistic and translational studies, this platform will enable disease-relevant modelling of PI3K-driven vascular malformations, including PIK3CA -related overgrowth spectrum (PROS) and PTEN hamartoma tumour syndrome (PHTS).

Emerging evidence suggests that Parkinson’s disease (PD) may have its origin in the enteric nervous system (ENS), from where α-synuclein (αS) pathology spreads to the brain1–4. Decades before the onset of motor symptoms, patients with PD suffer from constipation and present with circulating T cells responsive to αS, suggesting that peripheral immune responses initiated in the ENS may be involved in the early stages of PD1,5–7. However, cellular mechanisms that trigger αS pathology in the ENS and its spread along the gut–brain axis remain elusive. Here we demonstrate that muscularis macrophages (ME-Macs), housekeepers of ENS integrity and intestinal homeostasis, modulate αS pathology and neurodegeneration in models of PD8,9. ME-Macs contain misfolded αS, adopt a signature reflecting endolysosomal dysfunction and modulate the expansion of T cells that travel from the ENS to the brain through the dura mater as αS pathology progresses. Directed ME-Mac depletion leads to reduced αS pathology in the ENS and central nervous system, prevents T cell expansion and mitigates neurodegeneration and motor dysfunction, suggesting a role for ME-Macs as early cellular initiators of αS pathology along the gut–brain axis. Understanding these mechanisms could pave the way for early-stage biomarkers in PD.

Signaling receptors often encounter multiple ligands and have been shown to respond selectively to generate appropriate, context-specific outcomes. At thermal equilibrium, ligand specificity is limited by the relative affinities of ligands for their receptors. Here, we present a non-equilibrium model in which receptors overcome thermodynamic constraints to preferentially signal from specific ligands while suppressing others. In our model, multi-site phosphorylation and active receptor degradation act in concert to regulate ligand specificity, with receptor degradation, a common motif in eukaryotes, providing a previously under-appreciated layer of control. Here, ligand-bound receptors undergo sequential phosphorylation, with progression restarted by ligand unbinding or receptor turnover. High-affinity complexes are kinetically sorted toward degradation-prone states, while low-affinity complexes are sorted toward inactivated states, both limiting signaling. As a result, network activity is maximized for ligands with intermediate affinities. This mechanism explains paradoxical experimental observations in receptor tyrosine kinase signaling, including non-monotonic dependence of signaling output on ligand affinity and kinase activity. Given the ubiquity of multi-site phosphorylation and ligand-induced degradation across signaling receptors, we propose that kinetic sorting may be a general non-equilibrium ligand-discrimination strategy used by multiple signaling receptors.

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is unclear. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 is required for lysophagic flux after lysosomal damage but is not involved in the initial phagophore formation on terminally damaged lysosomes. Instead, ATXN3 is recruited to a distinct subset of lysosomes that are decorated with phosphatidylinositol-(4,5)-bisphosphate and that are not yet fully reacidified. There, ATXN3, along with its partner VCP/p97, targets and turns over K48-K63-branched ubiquitin conjugates. ATXN3 thus facilitates degradation of a fraction of LAMP2 via microautophagy to regenerate the lysosomal membrane and to thereby reestablish degradative capacity needed also for completion of lysophagy. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

Targeted protein degradation (TPD) destroys proteins of interest (POIs) by hijacking the cellular proteolytic machinery. Most proteins in cells exist and function as part of multi-protein or macromolecular complexes, thereby allowing a single protein to control multiple biological processes. Therefore, when a small molecule degrader induces proximity between an E3 ligase and the POI, the macromolecular context of the POI potentially influences the degradation outcomes of the POI and of the complex components. Here, we explore degradation of the eight CK1α-SACK1(A-H) (formerly known as FAM83A-H) complexes initiated by molecular glue degraders primarily designed to target Ser/Thr kinase CK1α. We demonstrate that lenalidomide-derived degraders DEG-77 and SJ3149, which selectively target the CK1α isoform, co-degrade multiple SACK1(A-H) proteins. We show that the degradation of SACK1(A-H) proteins by DEG-77 and SJ3149 requires CK1α, the CUL4ACRBN E3 ligase complex and the proteasome. In cells derived from palmoplantar keratoderma patients harbouring the CK1α-binding deficient SACK1GR265P mutation, DEG-77 targets CK1α and mitotic SACK1D but not SACK1GR265P, highlighting the requirement for CK1α-SACK1(A-H) interaction to achieve co-degradation. Our study underscores the importance of POI context in TPD and reinforces the potential for selectively targeting specific protein complexes for degradation.

Mutations in Leucine-Rich Repeat protein Kinase 2 (LRRK2) are associated with Parkinson’s disease (PD) and Crohn’s disease (CD), but the regulation of LRRK2 during inflammation remains relatively unexplored. Here we describe the development of a flow cytometry-based assay to assess LRRK2 activity in individual cells and the generation of an EGFP-Lrrk2 knock-in reporter mouse to analyse cell-specific LRRK2 expression. Using these tools, we measured LRRK2 levels and activity in murine splenic and intestinal immune cells and in human blood. Anti-CD3 induced inflammation increases LRRK2 expression and activity in B cells and monocytes, while in mature neutrophils, inflammation stimulates activity but reduces LRRK2 expression. A kinase-activating PD-associated LRRK2-R1441C mutation exacerbates inflammation-induced activation of LRRK2 specifically in monocytes and macrophages. We identify IL-4 as a novel T-cell-derived factor that upregulates LRRK2 expression and activity in B cells, replicating inflammatory effects observed in vivo. Our findings provide valuable new insights into the regulation of the LRRK2 pathway in immune cells, crucial for understanding LRRK2 and its therapeutic potential in inflammatory diseases such as CD.