Biology subjects

Sathe, G.

Publications and source records attributed to Sathe, G..

10 recordsLinked to original sources

bioRxiv2026

Targeted dephosphorylation of Tau at the endogenous level promotes its association with microtubules.

Hyperphosphorylation of Tau is a contributory factor for Tauopathies, which constitute a spectrum of neurodegenerative disorders including Alzheimer's disease (AD). Inhibitors of Tau kinases or activators of the Tau phosphatase PP2A to reverse Tau hyperphosphorylation have shown limited therapeutic promise, due primarily to a lack of Tau selectivity. An innovative strategy to potentially circumvent these limitations would be to selectively target the dephosphorylation of phospho-residues on Tau. In this study, we provide evidence for targeted dephosphorylation of phospho-Tau at the endogenous level, including those known to promote Tau aggregation. By recruiting PPP1CA and PPP2CA Affinity-directed (Ad)-Phosphatases to Tau, we demonstrate highly selective targeted dephosphorylation of multiple phospho-residues on Tau in SK-N-MC neuroblastoma cells, leading to its stabilization in microtubules. Moreover, by using a heterobifunctional molecule termed BDPIC (bromoTAG-dTAG proximity-inducing chimera) in cells harbouring bromoTAG and dTAG knockins on Tau and PPP2CA, respectively, we demonstrate highly selective dephosphorylation phospho-residues on Tau.

neuroscience↗

bioRxiv2026

Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

biochemistry↗

bioRxiv2026

Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

Medulloblastoma, the most common malignant pediatric brain tumor, arises from developmental aberrations of cerebellar precursor cells. The CUL3-RING ubiquitin ligase adaptor KBTBD4 is recurrently mutated in medulloblastoma subgroups 3 and 4. While KBTBD4 mutations confer a gain-of-function phenotype leading to aberrant degradation of transcriptional repressors, endogenous targets of this E3 ligase remain unknown. Here, we identify the PP2A-A scaffolding subunit of the PP2A phosphatase as a CRL3KBTBD4 substrate. Using a combination of proteomics, cell biology, biochemical reconstitution, and cryo-EM structural analyses, we show that CRL3KBTBD4 mediates orphan quality control by targeting free PP2A-A for degradation to safeguard phosphatase activity. Loss of KBTBD4 or its mutation in medulloblastoma cause PP2A-A accumulation, hence affecting phospho-dependent signaling pathways in cancer development. Disease mutations in KBTBD4 thus elicit a dual phenotype: gain-of-function degradation of transcriptional repressors combined with loss of PP2A quality control, which dysregulates multiple signaling events implicated in cancer, including telomere length regulation.

cell biology↗

bioRxiv2025

Linker-rigidified VHL homodimerizers convert degraders into stabilizers of non-ubiquitinable ternary complexes

Protein degraders recruit E3 ligases to targets for ubiquitination and subsequent proteasomal degradation. Efficient degradation typically correlates with long-lived ternary complexes that position target lysines for productive ubiquitination. Here, we determine a cryo-EM structure of the Cullin 2 RING VHL (CRL2VHL) ligase dimerized by the VHL homo-PROTAC CM11, wherein one CRLVHL acts as the ligase and a second VHL as the neo-substrate. Guided by this structure, we design side-by-side, linker-rigidified VHL homodimerizers that bias the relative VHL orientation away from the E2[~]Ub active site, contrasting the flexible, head-to-head PEG linkage of CM11. Biophysical binding and in vitro ubiquitination assays show that these compounds form stable, long-lived and compact ternary complexes that are incompatible with VHL cross-ubiquitination. In cells, the compounds stabilize VHL and concomitantly inhibit it to elevate HIF-1 levels. Thus, a stable ternary complex can be non-productive for ubiquitination, and linker architecture can reprogram degraders into stabilizers by controlling target ubiquitinability.

biochemistry↗

bioRxiv2025

Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design

Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTACs) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen Receptor (ER) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ER, promoting cooperative complex formation and target ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovers de novo VHL-14-3-3{zeta} contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes.

biochemistry↗

bioRxiv2025

Dual E3 ligase recruitment by monovalent degraders enables redundant and tuneable degradation of SMARCA2/4

Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compounds "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.

biochemistry↗

bioRxiv2025

Data-independent acquisition (DIA) approach for comprehensive ubiquitinome profiling in targeted protein degradation

Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a cutting-edge workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly precursors corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins with various TPD approaches. Therefore, our workflow holds tremendous potential for rapidly establishing mode of action for various TPD modalities, including PROTACs and molecular glues.

cell biology↗

bioRxiv2025

Targeted degradation of α-synuclein prevents PFF-induced aggregation

Accumulation of misfolded -synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinsons Disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Therefore, clearance of misfolded -synuclein from dopaminergic neurons could in principle offer a therapeutic window for Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human -synuclein protein and demonstrate targeted degradation of endogenous -synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of -synuclein prevents the pre-formed fibril (PFF)-induced aggregation of -synuclein in primary neurons derived from rats expressing human -synuclein. This approach represents the first demonstration of nanobody-guided proteasomal degradation of all clinically relevant -synuclein variants, highlighting its potential as a therapeutic strategy against Synucleinopathies.

neuroscience↗

bioRxiv2024

AMPK activation promotes transcriptional activation of TFEB through its dephosphorylation

Transcription Factor EB (TFEB) is a critical regulator of lysosomal biogenesis, autophagy and energy homeostasis through controlling expression of genes belonging to the coordinated lysosomal expression and regulation network. AMP-activated protein kinase (AMPK) has been reported to phosphorylate TFEB at three conserved C-terminal serine residues (S466, S467, S469) and these phosphorylation events were essential for transcriptional activation of TFEB. In sharp contrast to this proposition, here we demonstrate that AMPK activation leads to dephosphorylation of the C-terminal sites, and that AMPK is dispensable for mTORC1-mediated/torin1-sensitive TFEB activation. We show that a synthetic peptide encompassing C-terminal serine residues of TFEB is a poor substrate of AMPK. Treatment of cells with AMPK activator (MK-8722) or mTOR inhibitor (torin1) robustly dephosphorylated TFEB not only at mTORC1-targeted N-terminal serine sites, but also at the C-terminal sites. Loss of function of AMPK abrogated MK-8722-but not torin1-induced dephosphorylation and induction of the vast majority of TFEB target genes.

biochemistry↗

bioRxiv2023

Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase that regulates a plethora of cellular processes. PP2A operates as a holoenzyme complex, comprising one each of the scaffolding (A), regulatory (B) and catalytic (C) subunits. PPP2CA is the principal catalytic subunit of the PP2A holoenzyme complex. Although previous studies have reported many substrates of specific PP2A holoenzyme complexes, the full scope of PP2A substrates in cells remains to be defined. To address this, we generated HEK293 cells in which PPP2CA was homozygously knocked in with a dTAG, allowing for efficient and selective degradation of dTAG-PPP2CA with proteolysis-targeting chimeras (PROTACs) targeting the dTAG. By employing an unbiased global phospho-proteomic analysis, we identified 6,280 phospho-peptides corresponding to 2,204 proteins that showed a significant increase in abundance upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. Among these, some were established PP2A substrates, while most were novel. Bioinformatic analyses revealed the involvement of the identified potential PPP2CA substrates in many cellular processes, including spliceosome function, the cell cycle, RNA transport and ubiquitin-mediated proteolysis. We show that a pSP/pTP motif is a predominant target for PPP2CA. We confirmed some of our phospho-proteomic data with immunoblotting, by utilising commercially available phospho-specific antibodies. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.

cell biology↗