bims-proteo 2024-12-22 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–12–22
thirty-two papers selected by
Eric Chevet, INSERM

Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex.
Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004.
eEF2K regulates pain through translational control of BDNF.
Redirecting E3 Ubiquitin Ligases for Targeted Protein Degradation with Heterologous Recognition Domains.
Drug Discovery Approaches to Target E3 Ligases.
Mechanism of small heat shock protein client sequestration and induced polydispersity.
DARPins as a novel tool to detect and degrade p73.
Dynamic modulation of IRE1α-XBP1 signaling by adenovirus.
A nuclear protein quality control system for elimination of nucleolus-related inclusions.
Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.
Analyses of translation factors Dbp1 and Ded1 reveal the cellular response to heat stress to be separable from stress granule formation.
Harnessing the FBXW7 somatic mutant R465C for targeted protein degradation.
Mitochondrial unfolded protein response-dependent β-catenin signaling promotes neuroendocrine prostate cancer.
Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in charcot-marie-tooth type 2 neuropathies.
BindCraft: one-shot design of functional protein binders.
The UFMylation pathway is impaired in Alzheimer's disease.
A proteome-wide yeast degron collection for the dynamic study of protein function.
CKAP4 in hepatocellular carcinoma: competitive RETREG1/FAM134B binding, reticulophagy regulation, and cancer progression.
Temperature perception by ER UPR promotes preventive innate immunity and longevity.
Molecular Insights into the Regulation of GNPTAB by TMEM251.
Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress.
Proteasome Activators and Ageing: Restoring Proteostasis Using Small Molecules.
GSK3β phosphorylation catalyzes the aggregation of tau into Alzheimer's disease-like filaments.
TXNDC15, an ER-localized thioredoxin-like transmembrane protein, contributes to ciliary transition zone integrity.
The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance.
Deep representation learning of protein-protein interaction networks for enhanced pattern discovery.
Macronucleophagy maintains cell viability under nitrogen starvation by modulating micronucleophagy.
Proteomics analysis reveals the differential impact of the p97 inhibitor CB-5083 on protein levels in various cellular compartments of the HL-60 cell line.
Disrupted uromodulin trafficking is rescued by targeting TMED cargo receptors.
Protocol to study secretome interactions using extracellular proximity labeling.
Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice.
ZDHHC20 mediated S-palmitoylation of fatty acid synthase (FASN) promotes hepatocarcinogenesis.

Commun Biol. 2024 Dec 19. 7(1): 1668

Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex.

Matthew E R Maitland, Gabriel Onea, Dominic D G Owens, Brianna C Gonga-Cavé, Xu Wang, Cheryl H Arrowsmith, Dalia Barsyte-Lovejoy, Gilles A Lajoie, Caroline Schild-Poulter.

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.

DOI: https://doi.org/10.1038/s42003-024-07371-3
bioRxiv. 2024 Dec 07. pii: 2024.12.07.627262. [Epub ahead of print]

Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004.

Phoebe H Fechtmeyer, Johannes T-H Yeh.

  Targeted protein degradation (TPD) is a promising new therapeutic modality that leverages the endogenous cellular protein degradation machinery of the ubiquitin-proteasome system (UPS) to degrade selected proteins. Recently, we developed a synthetic macrocycle ligand to recruit CUL3KLHL20 E3 ligase for TPD. Using this KLHL20 ligand, we constructed the PROTAC BTR2004, which demonstrated potent degradation of BET family proteins BRD 2, 3, and 4. As the TPD field expands, it is important to understand the cellular and biochemical properties of all utilized E3 ligases. Herein we report the temporal and spatial processes of BTR2004-facilitated BET family protein degradation by KLHL20: The target protein degradation kinetics, BTR2004 intracellular activity half-life, and the onset of BTR2004 cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the subcellular location of the ternary complex assembly upon BTR2004 treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated when designing future PROTAC molecules.

Keywords:  CUL3; E3 ligase; KLHL20; PROTAC; macrocycle; targeted protein degradation

DOI:  https://doi.org/10.1101/2024.12.07.627262
Mol Cell. 2024 Dec 14. pii: S1097-2765(24)00948-1. [Epub ahead of print]

eEF2K regulates pain through translational control of BDNF.

Patrick R Smith, Guadalupe Garcia, Angela R Meyer, Alexey G Ryazanov, Tao Ma, Sarah Loerch, Zachary T Campbell.

  mRNA translation is integral to pain, yet the key regulatory factors and their target mRNAs are unclear. Here, we uncover a mechanism that bridges noxious insults to multiple phases of translational control in murine sensory neurons. We find that a painful cue triggers repression of peptide chain elongation through activation of elongation factor 2 kinase (eEF2K). Attenuated elongation is sensed by a ribosome-coupled mechanism that triggers the integrated stress response (ISR). Both eEF2K and the ISR are required for pain-associated behaviors in vivo. This pathway simultaneously induces biosynthesis of brain-derived neurotrophic factor (BDNF). Selective blockade of Bdnf translation has analgesic effects in vivo. Our data suggest that precise spatiotemporal regulation of Bdnf translation is critical for appropriate behavioral responses to painful stimuli. Overall, our results demonstrate that eEF2K resides at the nexus of an intricate regulatory network that links painful cues to multiple layers of translational control.

Keywords:  BDNF; GCN2; P-stalk; eEF2K; eIF2α; integrated stress response; pain; selective translation

DOI:  https://doi.org/10.1016/j.molcel.2024.11.023
J Biol Chem. 2024 Dec 13. pii: S0021-9258(24)02579-1. [Epub ahead of print] 108077

Redirecting E3 Ubiquitin Ligases for Targeted Protein Degradation with Heterologous Recognition Domains.

Huan Yang, Ge Zheng, Grace Y Li, Alia Alshaye, Stuart H Orkin.

  Targeted protein degradation (TPD) mediated by PROTACs (proteolysis targeting chimeras) or molecular glues is an emerging therapeutic strategy. Despite greater than 600 E3 ligases and their associated components, a limited number have been deployed in TPD. Those commonly used include cereblon (CRBN) and von Hippel-Lindau tumor suppressor (VHL), which are expressed widely and for which high affinity ligands are available. Limiting TPD to specific cells or tissues would be desirable in many settings. To this goal we have determined the potential of two erythroid cell-enriched E3 ligases, TRIM10 and TRIM58, to degrade a protein of interest, BCL11A, a validated therapeutic target for the β-hemoglobinopathies. We established a general strategy in which heterologous recognition domains replace the PRY-SPRY domain of TRIM10 and TRIM58. Recruitment of TRIM10 or TRIM58 to BCL11A by coiled-coil peptides, nanobodies, or the substrate recognition domain of CRBN led to its degradation. Our findings illustrate a strategy that may be widely useful in evaluating the TPD potential of other E3 ubiquitin ligases and provide a rationale for discovery of ligands for TRIM10 and TRIM58 for erythroid-selective depletion of proteins of interest.

Keywords:  BCL11A; E3 Ubiquitin Ligases; PROTACs; TRIM10; TRIM58; targeted protein degradation

DOI:  https://doi.org/10.1016/j.jbc.2024.108077
Chembiochem. 2024 Dec 17. e202400656

Drug Discovery Approaches to Target E3 Ligases.

Alejandra Rodríguez-Gimeno, Carles Galdeano.

  Targeting E3 ligases is a challenging area in drug discovery. Despite the human genome encoding for more than 600 E3 ubiquitin ligases, only a handful of E3 ligases have been pharmacologically modulated or exploited for targeted protein degradation (TPD) strategies. The main obstacle for hijacking these E3 ligases is the lack of small-molecule ligands. As research into this field advances, the identification of new small molecules capable of binding to E3 ligases has become an essential pursuit. These ligases not only expand the repertoire of druggable targets but also offer the potential for increased specificity and selectivity in protein degradation. The synergy between academia and industry is key, as it combines academic expertise in fundamental research with the industrial capabilities of translating these findings into novel therapeutics. In this review, we provide an overview of the different strategies employed in academia and industry to the discovery of new E3 ligases ligands, showing them with illustrative cases.

Keywords:  E3 ligases; PROTACs; Screening technologies; Targeted protein degradation; Undruggable

DOI:  https://doi.org/10.1002/cbic.202400656
bioRxiv. 2024 Dec 06. pii: 2024.12.03.626640. [Epub ahead of print]

Mechanism of small heat shock protein client sequestration and induced polydispersity.

Adam P Miller, Steve L Reichow.

Small heat shock proteins (sHSPs) act as first responders during cellular stress by recognizing and sequestering destabilized proteins (clients), preventing their aggregation and facilitating downstream refolding or degradation 1-3 . This chaperone function is critically important to proteostasis, conserved across all kingdoms of life, and associated with various protein misfolding diseases in humans 4,5 . Mechanistic insights into how sHSPs sequester destabilized clients have been limited due to the extreme molecular plasticity and client-induced polydispersity of sHSP/client complexes 6-8 . Here, we present high-resolution cryo-EM structures of the sHSP from Methanocaldococcus jannaschii ( mj HSP16.5) in both the apo-state and in an ensemble of client-bound states. The ensemble not only reveals key molecular mechanisms by which sHSPs respond to and sequester client proteins, but also provides insights into the cooperative nature of chaperone-client interactions. Engagement with destabilized client induces a polarization of stability across the mj HSP16.5 scaffold, proposed to facilitate higher-order assembly and enhance client sequestration capacity. Some higher-order sHSP oligomers appear to form through simple insertion of dimeric subunits into new geometrical features, while other higher-order states suggest multiple sHSP/client assembly pathways. Together, these results provide long-sought insights into the chaperone function of sHSPs and highlight the relationship between polydispersity and client sequestration under stress conditions.

DOI: https://doi.org/10.1101/2024.12.03.626640
Cell Death Dis. 2024 Dec 18. 15(12): 909

DARPins as a novel tool to detect and degrade p73.

Philipp Münick, Jasmin Zielinski, Alexander Strubel, Niklas Gutfreund, Birgit Dreier, Jonas V Schaefer, Birgit Schäfer, Jakob Gebel, Christian Osterburg, Apirat Chaikuad, Stefan Knapp, Andreas Plückthun, Volker Dötsch.

The concept of Targeted Protein Degradation (TPD) has been introduced as an attractive alternative to the development of classical inhibitors. TPD can extend the range of proteins that can be pharmacologically targeted beyond the classical targets for small molecule inhibitors, as a binding pocket is required but its occupancy does not need to lead to inhibition. The method is based on either small molecules that simultaneously bind to a protein of interest and to a cellular E3 ligase and bring them in close proximity (molecular glue) or a bi-functional molecule synthesized from the chemical linkage of a target protein-specific small molecule and one that binds to an E3 ligase (Proteolysis Targeting Chimeras (PROTAC)). The further extension of this approach to bioPROTACs, in which a small protein-based binding module is fused directly to an E3 ligase or an E3 ligase adaptor protein, makes virtually all proteins amenable to targeted degradation, as this method eliminates the requirement for binding pockets for small molecules. Designed Ankyrin Repeat Proteins (DARPins) represent a very attractive class of small protein-based binding modules that can be used for the development of bioPTOTACS. Here we describe the characterization of two DARPins generated against the oligomerization domain and the SAM domain of the transcription factor p73, a member of the p53 protein family. The DARPins can be used for (isoform-)selective pulldown experiments both in cell culture as well as primary tissue lysates. We also demonstrate that they can be used for staining in cell culture experiments. Fusing them to the speckle type POZ protein (SPOP), an adaptor protein for cullin-3 E3 ligase complexes, yields highly selective and effective degraders. We demonstrate that selective degradation of the ΔNp73α isoform reactivates p53.

DOI: https://doi.org/10.1038/s41419-024-07304-2
bioRxiv. 2024 Dec 05. pii: 2024.11.30.626188. [Epub ahead of print]

Dynamic modulation of IRE1α-XBP1 signaling by adenovirus.

Yumi Jang, Fred Bunz.

The abundant production of foreign proteins and nucleic acids during viral infection elicits a variety of stress responses in host cells. Viral proteins that accumulate in the endoplasmic reticulum (ER) can trigger the unfolded protein response (UPR), a coordinated signaling program that culminates in the expression of downstream genes that collectively restore protein homeostasis. The model pathogen adenovirus serotype 5 (HAdV5) activates the UPR via the signaling axis formed by inositol-requiring enzyme type 1 (IRE1α) and the X-box binding protein 1 (XBP1), a transcription factor required for immune function. Recent studies have suggested that IRE1α-XBP1 activity supports adenovirus replication. Here, we show that HAdV5 exerted opposing effects on IRE1α and XBP1. IRE1α was activated in response to HAdV5 but the production of the XBP1 isoform, XBP1s, was post-transcriptionally blocked. The tumor suppressor p53, which is eliminated by HAdV5 after infection, inhibited IRE1α activation. The de-repression of IRE1α following the degradation of p53 conceivably reflects a novel antiviral mechanism, which HAdV5 ultimately evades by suppressing XBP1s. Our findings highlight the defective antiviral defenses in cancer cells and further illustrate the opposing mechanisms used by adenoviruses and their host cells to exert control over the UPR, a critical determinant of cell fate.

DOI: https://doi.org/10.1101/2024.11.30.626188
EMBO J. 2024 Dec 17.

A nuclear protein quality control system for elimination of nucleolus-related inclusions.

Lorène Brunello, Jolanta Polanowska, Léo Le Tareau, Chantal Maghames, Virginie Georget, Charlotte Guette, Karima Chaoui, Stéphanie Balor, Marie-Françoise O'Donohue, Marie-Pierre Bousquet, Pierre-Emmanuel Gleizes, Dimitris P Xirodimas.

  The identification of pathways that control elimination of protein inclusions is essential to understand the cellular response to proteotoxicity, particularly in the nuclear compartment, for which our knowledge is limited. We report that stress-induced nuclear inclusions related to the nucleolus are eliminated upon stress alleviation during the recovery period. This process is independent of autophagy/lysosome and CRM1-mediated nuclear export pathways, but strictly depends on the ubiquitin-activating E1 enzyme, UBA1, and on nuclear proteasomes that are recruited into the formed inclusions. UBA1 activity is essential only for the recovery process but dispensable for nuclear inclusion formation. Furthermore, the E3 ligase HUWE1 and HSP70 are components of the ubiquitin/chaperone systems that promote inclusion elimination. The recovery process also requires RNA Pol I-dependent production of the lncRNA IGS42 during stress. IGS42 localises within the formed inclusions and promotes their elimination by preserving the mobility of resident proteins. These findings reveal a protein quality control system that operates within the nucleus for the elimination of stress-induced nucleolus-related inclusions.

Keywords:  IGS ncRNAs; Nucleolus; Proteasome; Proteotoxic Stress; Ubiquitin/NEDD8

DOI:  https://doi.org/10.1038/s44318-024-00333-9
Autophagy. 2024 Dec 18.

Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation.

Van Bui, Xinwen Liang, Yansheng Ye, William Giang, Fang Tian, Yoshinori Takahashi, Hong-Gang Wang.

  Macroautophagy/autophagy, an evolutionarily conserved cellular degradation pathway, involves phagophores that sequester cytoplasmic constituents and mature into autophagosomes for subsequent lysosomal delivery. The ATG8 gene family, comprising the MAP1LC3/LC3 and GABARAP/GBR subfamilies in mammals, encodes ubiquitin-like proteins that are conjugated to phagophore membranes during autophagosome biogenesis. A central question in the field is how Atg8-family proteins are precisely involved in autophagosome formation, which remains controversial and challenging, at least in part due to the short lifespan of phagophores. In this study, we depleted the autophagosome closure regulator VPS37A to arrest autophagy at the vesicle completion step and determined the roles of mammalian Atg8-family proteins (mATG8s) in nutrient starvation-induced autophagosome biogenesis. Our investigation revealed that LC3 loss hinders phagophore formation, while GBR loss impedes both phagophore formation and expansion. The defect in membrane expansion by GBR loss appears to be attributed to compromised recruitment of ATG proteins containing an LC3-interacting region (LIR), including ULK1 and ATG3. Moreover, a combined deficiency of both LC3 and GBR subfamilies nearly completely inhibits phagophore formation, highlighting their redundant regulation of this process. Consequently, cells lacking all mATG8 members exhibit defects in downstream events such as ESCRT recruitment and autophagic flux. Collectively, these findings underscore the critical roles of mammalian Atg8-family proteins in phagophore formation and expansion during autophagy.

Keywords:  Autophagosome closure; ESCRT; LIR; The mammalian Atg8 family of proteins; membrane expansion; phagophore formation

DOI:  https://doi.org/10.1080/15548627.2024.2443300
Cell Rep. 2024 Dec 13. pii: S2211-1247(24)01410-4. [Epub ahead of print]43(12): 115059

Analyses of translation factors Dbp1 and Ded1 reveal the cellular response to heat stress to be separable from stress granule formation.

Naohiro Kuwayama, Emily Nicole Powers, Matej Siketanc, Camila Ines Sousa, Kendra Reynaud, Marko Jovanovic, Maria Hondele, Nicholas Thomas Ingolia, Gloria Ann Brar.

  Ded1 and Dbp1 are paralogous conserved DEAD-box ATPases involved in translation initiation in yeast. In long-term starvation states, Dbp1 expression increases and Ded1 decreases, whereas in cycling mitotic cells, Dbp1 is absent. Inserting DBP1 in place of DED1 cannot replace Ded1 function in supporting mitotic translation, partly due to inefficient translation of the DBP1 coding region. Global translation measurements, activity of mRNA-tethered proteins, and growth assays show that-even at matched protein levels-Ded1 is better than Dbp1 at activating translation, especially for mRNAs with structured 5' leaders. Heat-stressed cells normally downregulate translation of structured housekeeping transcripts and halt growth, but neither occurs in Dbp1-expressing cells. This failure to halt growth in response to heat is not based on deficient stress granule formation or failure to reduce bulk translation. Rather, it depends on heat-triggered loss of Ded1 function mediated by an 11-amino-acid interval within its intrinsically disordered C terminus.

Keywords:  CP: Molecular biology; DDX3; DEAD-box helicase; Dbp1; Ded1; Translation; heat stress; ribosome profiling; stress granules; translation initiation

DOI:  https://doi.org/10.1016/j.celrep.2024.115059
bioRxiv. 2024 Dec 04. pii: 2024.12.03.626601. [Epub ahead of print]

Harnessing the FBXW7 somatic mutant R465C for targeted protein degradation.

Ananya A Basu, Chenlu Zhang, Milad Rouhimoghadam, Anil Vasudevan, Justin M Reitsma, Xiaoyu Zhang.

Targeted protein degradation (TPD) is a pharmacological strategy that eliminates specific proteins from cells by harnessing cellular proteolytic degradation machinery. In proteasome-dependent TPD, expanding the repertoire of E3 ligases compatible with this approach could enhance the applicability of this strategy across various biological contexts. In this study, we discovered that a somatic mutant of FBXW7, R465C, can be exploited by heterobifunctional compounds for targeted protein degradation. This work demonstrates the potential of utilizing mutant E3 ligases that occur exclusively in diseased cells for TPD applications.

DOI: https://doi.org/10.1101/2024.12.03.626601
Oncogene. 2024 Dec 17.

Mitochondrial unfolded protein response-dependent β-catenin signaling promotes neuroendocrine prostate cancer.

Jordan Alyse Woytash, Rahul Kumar, Ajay K Chaudhary, Cullan Donnelly, Adam Wojtulski, Murali Bethu, Jianmin Wang, Joseph Spernyak, Peter Bross, Neelu Yadav, Joseph R Inigo, Dhyan Chandra.

The mitochondrial unfolded protein response (UPRmt) maintains mitochondrial quality control and proteostasis under stress conditions. However, the role of UPRmt in aggressive and resistant prostate cancer is not clearly defined. We show that castration-resistant neuroendocrine prostate cancer (CRPC-NE) harbored highly dysfunctional oxidative phosphorylation (OXPHOS) Complexes. However, biochemical and protein analyses of CRPC-NE tumors showed upregulation of nuclear-encoded OXPHOS proteins and UPRmt in this lethal subset of prostate cancer suggestive of compensatory upregulation of stress signaling. Genetic deletion and pharmacological inhibition of the main chaperone of UPRmt heat shock protein 60 (HSP60) reduced neuroendocrine prostate cancer (NEPC) growth in vivo as well as reverted NEPC cells to a more epithelial-like state. HSP60-dependent aggressive NEPC phenotypes was associated with upregulation of β-catenin signaling both in cancer cells and in vivo tumors. HSP60 expression rendered enrichment of aggressive prostate cancer signatures and metastatic potential were inhibited upon suppression of UPRmt. We discovered that UPRmt promoted OXPHOS functions including mitochondrial bioenergetics in CRPC-NE via regulation of β-catenin signaling. Mitochondrial biogenesis facilitated cisplatin resistance and inhibition of UPRmt resensitizes CRPC-NE cells to cisplatin. Together, our findings demonstrated that UPRmt promotes mitochondrial health via upregulating β-catenin signaling and UPRmt represents viable therapeutic target for NEPC.

DOI: https://doi.org/10.1038/s41388-024-03261-4
Autophagy. 2024 Dec 19.

Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in charcot-marie-tooth type 2 neuropathies.

Angela Sisto, Tamira van Wermeskerken, Michael Pancher, Pamela Gatto, Bob Asselbergh, Ágata Sofia Assunção Carreira, Vicky De Winter, Valentina Adami, Alessandro Provenzani, Vincent Timmerman.

  HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1P182L and HSPB8K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies.

Keywords:  Autophagy inducer; drug repurposing; inherited peripheral neuropathy; motor neurons; phenotypical screening; small heat shock proteins

DOI:  https://doi.org/10.1080/15548627.2024.2439649
bioRxiv. 2024 Dec 07. pii: 2024.09.30.615802. [Epub ahead of print]

BindCraft: one-shot design of functional protein binders.

Martin Pacesa, Lennart Nickel, Christian Schellhaas, Joseph Schmidt, Ekaterina Pyatova, Lucas Kissling, Patrick Barendse, Jagrity Choudhury, Srajan Kapoor, Ana Alcaraz-Serna, Yehlin Cho, Kourosh H Ghamary, Laura Vinué, Brahm J Yachnin, Andrew M Wollacott, Stephen Buckley, Adrie H Westphal, Simon Lindhoud, Sandrine Georgeon, Casper A Goverde, Georgios N Hatzopoulos, Pierre Gönczy, Yannick D Muller, Gerald Schwank, Daan C Swarts, Alex J Vecchio, Bernard L Schneider, Sergey Ovchinnikov, Bruno E Correia.

Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 1 to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins, and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity, and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Lastly, we utilize cell surface receptor-specific binders to redirect AAV capsids for targeted gene delivery. This work represents a significant advancement towards a "one design-one binder" approach in computational design, with immense potential in therapeutics, diagnostics, and biotechnology.

DOI: https://doi.org/10.1101/2024.09.30.615802
Mol Neurodegener. 2024 Dec 18. 19(1): 97

The UFMylation pathway is impaired in Alzheimer's disease.

Tingxiang Yan, Michael G Heckman, Emily C Craver, Chia-Chen Liu, Bailey D Rawlinson, Xue Wang, Melissa E Murray, Dennis W Dickson, Nilufer Ertekin-Taner, Zhenkun Lou, Guojun Bu, Wolfdieter Springer, Fabienne C Fiesel.

   BACKGROUND: Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles made of hyperphosphorylated tau and senile plaques composed of beta-amyloid. These pathognomonic deposits have been implicated in the pathogenesis, although the molecular mechanisms and consequences remain undetermined. UFM1 is an important, but understudied ubiquitin-like protein that is covalently attached to substrates. UFMylation has recently been identified as major modifier of tau aggregation upon seeding in experimental models. However, potential alterations of the UFM1 pathway in human AD brain have not been investigated yet.
METHODS: Here we used frontal and temporal cortex samples from individuals with or without AD to measure the protein levels of the UFMylation pathway in human brain. We used multivariable regression analyses followed by Bonferroni correction for multiple testing to analyze associations of the UFMylation pathway with neuropathological characteristics, primary biochemical measurements of tau and additional biochemical markers from the same cases. We further studied associations of the UFMylation cascade with cellular stress pathways using Spearman correlations with bulk RNAseq expression data and functionally validated these interactions using gene-edited neurons that were generated by CRISPR-Cas9.
RESULTS: Compared to controls, human AD brain had increased protein levels of UFM1. Our data further indicates that this increase mainly reflects conjugated UFM1 indicating hyperUFMylation in AD. UFMylation was strongly correlated with pathological tau in both AD-affected brain regions. In addition, we found that the levels of conjugated UFM1 were negatively correlated with soluble levels of the deUFMylation enzyme UFSP2. Functional analysis of UFM1 and/or UFSP2 knockout neurons revealed that the DNA damage response as well as the unfolded protein response are perturbed by changes in neuronal UFM1 signaling.
CONCLUSIONS: There are marked changes in the UFMylation pathway in human AD brain. These changes are significantly associated with pathological tau, supporting the idea that the UFMylation cascade might indeed act as a modifier of tau pathology in human brain. Our study further nominates UFSP2 as an attractive target to reduce the hyperUFMylation observed in AD brain but also underscores the critical need to identify risks and benefits of manipulating the UFMylation pathway as potential therapeutic avenue for AD.

Keywords:  Alzheimer’s disease; Brain; Tau; UFM1; UFMylation; UFSP2

DOI:  https://doi.org/10.1186/s13024-024-00784-y
J Cell Biol. 2025 Feb 03. pii: e202409050. [Epub ahead of print]224(2):

A proteome-wide yeast degron collection for the dynamic study of protein function.

Rosario Valenti, Yotam David, Dunya Edilbi, Benjamin Dubreuil, Angela Boshnakovska, Yeynit Asraf, Tomer-Meir Salame, Ehud Sass, Peter Rehling, Maya Schuldiner.

Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations. We present the AID library which supplies "on demand" protein depletion to overcome these limitations. Here, each protein is tagged with a green fluorescent protein (GFP) and an auxin-inducible degron (AID), enabling rapid protein depletion that can be quantified systematically using the GFP element. We characterized the degradation response of all strains and demonstrated its utility by revisiting seminal yeast screens for genes involved in cell cycle progression as well as mitochondrial distribution and morphology. In addition to recapitulating known phenotypes, we also uncovered proteins with previously unrecognized roles in these central processes. Hence, our tool expands our knowledge of cellular biology and physiology by enabling access to phenotypes that are central to cellular physiology and therefore rapidly equilibrated.

DOI: https://doi.org/10.1083/jcb.202409050
Autophagy. 2024 Dec 17. 1-20

CKAP4 in hepatocellular carcinoma: competitive RETREG1/FAM134B binding, reticulophagy regulation, and cancer progression.

Jie Mo, Chen Su, Pengcheng Li, Zhenhua Yang, Ran Tao, Qiumeng Liu, Chaoyi Yuan, Lei Xu, Qianyun Ge, Deng Ning, Huifang Liang, Haidan Zhu, Yan Luo, Xiaoping Chen, Jin Chen, Bixiang Zhang.

  RETREG1/FAM134B is known for its role as a reticulophagy receptor. Our previous study established that RETREG1 is upregulated in hepatocellular carcinoma (HCC) and contributes to disease progression by activating the AKT signaling pathway. However, the specific mechanisms underlying the elevated expression of RETREG1 in HCC remain unclear. This study unveils the interaction of RETREG1 with CKAP4 and TRIM21. We demonstrated that TRIM21 ubiquitinates RETREG1 at K247 and K252, facilitating its proteasomal degradation. Conversely, CKAP4 shields RETREG1 from degradation by competitively binding to it, revealing a novel post-translational modification mechanism for RETREG1. By modulating RETREG1 expression, CKAP4, and TRIM21 intricately regulate reticulophagy. Additionally, we observed that stress-induced TRIM21 upregulation mitigates the function of RETREG1 to restore ER stress equilibrium. The oncogenic potential of CKAP4 in HCC was demonstrated using various animal models. Clinical sample analyses suggested that CKAP4 is a potential biomarker for HCC prognosis and diagnosis.Abbreviation: AKT: thymoma viral proto-oncogene; aa: amino acid; bp: base pair; CHX: cycloheximide; co-IP: co-Immunoprecipitation; CQ: chloroquine; CKAP4: cytoskeleton-associated protein 4; DKK1: dickkopf WNT signaling pathway inhibitor 1; DUBs: deubiquitinating enzymes; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HCC: hepatocellular carcinoma; HFD: high-fat diet; HiTV: hyperdynamic tail vein injection; IF: immunofluorescence; IHC: immunohistochemistry; IP-MS: immunoprecipitation-mass spectrometry; LIR: LC3-interacting region; mAbs: monoclonal antibodies; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mCherry: monomeric cherry; oe: overexpression; PDX: patient-derived tumor xenograft; reticulophagy: endoplasmic reticulum selective autophagy; RETREG1: reticulophagy regulator 1; RHD: reticulon-homology domain; Tg: thapsigargin; Tm: tunicamycin; TRIM21: tripartite motif-containing 21; UB: ubiquitin; WT: wild-type.

Keywords:  Autophagy; TRIM21; proteasome degradation; reticulophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2024.2435236
Cell Rep. 2024 Dec 14. pii: S2211-1247(24)01422-0. [Epub ahead of print]43(12): 115071

Temperature perception by ER UPR promotes preventive innate immunity and longevity.

Lei Zhou, Haoyu Zhuo, Jiaqi Jin, Anrui Pu, Qin Liu, Jiangbo Song, Xiaoling Tong, Haiqing Tang, Fangyin Dai.

  Microbial infectivity increases with rising environmental temperature, heightening the risk of infection to host organisms. The host's basal immunity is activated accordingly to mitigate upcoming pathogenic threats; still, how animals sense temperature elevation to adjust their preventive immune response remains elusive. This study reports that high temperature enhances innate immunity differently from pathogen infection. Unlike pathogen invasion requiring the mitochondrial unfolded protein response (UPR), high temperature engages the endoplasmic reticulum (ER) UPR to trigger the innate immune response. Furthermore, chronic activation of the XBP-1 UPR branch represses nucleolar ribosome biogenesis, a highly energy-consuming process, leading to lipid accumulation. The subsequent increase in oleic acid promotes the activation of the PMK-1 immune pathway. Additionally, ribosome biogenesis was identified as a regulator of longevity, wherein its impact is dependent on lipid metabolism and innate immunity. Collectively, our findings reveal the crucial role of ER-nucleolus crosstalk in shaping preventive immune responses and lifespan regulation.

Keywords:  C. elegans; CP: Cell biology; CP: Immunology; ER UPR; innate immunity; longevity; oleic acid; ribosome biogenesis; temperature

DOI:  https://doi.org/10.1016/j.celrep.2024.115071
bioRxiv. 2024 Dec 05. pii: 2024.12.05.627003. [Epub ahead of print]

Molecular Insights into the Regulation of GNPTAB by TMEM251.

Xi Yang, Balraj Doray, Varsha Venkatarangan, Benjamin C Jennings, Danielle Henn, Jiaxuan Liang, Haikun Zhao, Weichao Zhang, Bokai Zhang, Linchen Yu, Liang Chen, Stuart Kornfeld, Ming Li.

  In vertebrates, newly synthesized lysosomal enzymes traffick to lysosomes through the mannose-6-phosphate (M6P) pathway. The Golgi membrane protein TMEM251 was recently discovered to regulate lysosome biogenesis by controlling the level of GlcNAc-1-phosphotransferase (GNPT). However, its precise function remained unclear. In this study, we demonstrate that TMEM251 is a two-transmembrane protein indispensable for GNPT stability, cleavage by Site-1-Protease (S1P), and enzymatic activity. We reconcile conflicting models by showing that TMEM251 enhances GNPT cleavage and prevents its mislocalization to lysosomes for degradation. We further establish that TMEM251 achieves this by interacting with GOLPH3 and retromer complexes to anchor the TMEM251-GNPT complex at the Golgi. Alanine mutagenesis identified F4XXR7 motif in TMEM251's N-tail for GOLPH3 binding. Together, our findings uncover TMEM251's multi-faceted role in stabilizing GNPT, retaining it at the Golgi, and ensuring the fidelity of the M6P pathway, thereby providing insights into its molecular function.

Keywords:  GNPT; Golgi; MLII; MLV; Mucolipidosis; TMEM251/LYSET/GCAF; lysosome

DOI:  https://doi.org/10.1101/2024.12.05.627003
Elife. 2024 Dec 19. pii: RP96284. [Epub ahead of print]13

Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress.

Showkat Ahmad Dar, Sulochan Malla, Vlastimil Martinek, Matthew John Payea, Christopher Tai-Yi Lee, Jessica Martin, Aditya Jignesh Khandeshi, Jennifer L Martindale, Cedric Belair, Manolis Maragkakis.

  Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.

Keywords:  RNA decay; cell biology; cell line; genetics; genomics; human; mouse; stress response

DOI:  https://doi.org/10.7554/eLife.96284
Subcell Biochem. 2024 ;107 21-41

Proteasome Activators and Ageing: Restoring Proteostasis Using Small Molecules.

Arun Upadhyay, Vibhuti Joshi.

  Ageing is an inevitable phenomenon that remains under control of a plethora of signalling pathways and regulatory mechanisms. Slowing of cellular homeostasis and repair pathways, declining genomic and proteomic integrity, and deficient stress regulatory machinery may cause accumulating damage triggering initiation of pathways leading to ageing-associated changes. Multiple genetic studies in small laboratory organisms focused on the manipulation of proteasomal activities have shown promising results in delaying the age-related decline and improving the lifespan. In addition, a number of studies indicate a prominent role of small molecule-based proteasome activators showing positive results in ameliorating the stress conditions, protecting degenerating neurons, restoring cognitive functions, and extending life span of organisms. In this chapter, we provide a brief overview of the multi-enzyme proteasome complex, its structure, subunit composition and variety of cellular functions. We also highlight the strategies applied in the past to modulate the protein degradation efficiency of proteasome and their impact on rebalancing the proteostasis defects. Finally, we provide a descriptive account of proteasome activation mechanisms and small molecule-based strategies to improve the overall organismal health and delay the development of age-associated pathologies.

Keywords:  Ageing; Lifespan; Proteasome; Small molecules; Ubiquitin-proteasome system

DOI:  https://doi.org/10.1007/978-3-031-66768-8_2
Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2414176121

GSK3β phosphorylation catalyzes the aggregation of tau into Alzheimer's disease-like filaments.

Pijush Chakraborty, Alain Ibáñez de Opakua, Jeffrey A Purslow, Simon A Fromm, Debdeep Chatterjee, Milan Zachrdla, Shannon Zhuang, Sambhavi Puri, Benjamin Wolozin, Markus Zweckstetter.

  The pathological deposition of proteins is a hallmark of several devastating neurodegenerative diseases. These pathological deposits comprise aggregates of proteins that adopt distinct structures named strains. However, the molecular factors responsible for the formation of distinct aggregate strains are unknown. Here, we show that the serine/threonine kinase GSK3β catalyzes the aggregation of the protein tau into Alzheimer's disease (AD)-like filaments. We demonstrate that phosphorylation by GSK3β, but not by several other kinases, promotes the aggregation of full-length tau as well as enhances phase separation into gel-like condensate structures. Cryoelectron microscopy further reveals that the fibrils formed by GSK3β-phosphorylated tau adopt a fold comparable to that of paired helical filaments isolated from the brains of AD patients. Our results elucidate the intricate relationship between posttranslational modification and the formation of tau strains in neurodegenerative diseases.

Keywords:  Alzheimer's disease; NMR; cryo-EM; phosphorylation; tau

DOI:  https://doi.org/10.1073/pnas.2414176121
J Cell Sci. 2024 Dec 15. pii: jcs262123. [Epub ahead of print]137(24):

TXNDC15, an ER-localized thioredoxin-like transmembrane protein, contributes to ciliary transition zone integrity.

Shingo Yamazaki, Taiju Fujii, Shuhei Chiba, Hye-Won Shin, Kazuhisa Nakayama, Yohei Katoh.

  Primary cilia have specific proteins on their membrane to fulfill their sensory functions. Preservation of the specific protein composition of cilia relies on the barrier function of the transition zone (TZ) located at the ciliary base. Defects in cilia and the TZ cause ciliopathies, which have diverse clinical manifestations, including Meckel syndrome (MKS). Many of the proteins mutated in individuals with MKS are known to constitute the MKS module of the TZ. Although TXNDC15 (also known as MKS14) is a thioredoxin-related transmembrane protein that is localized mainly in the endoplasmic reticulum (ER) and is mutated in individuals with MKS, its role at the TZ or within cilia has not been characterized. Here, we show that TXNDC15-knockout cells have defects in MKS module assembly and in ciliary membrane protein localization. These defects in TXNDC15-knockout cells were not rescued by exogenous expression of any of the TXNDC15 constructs with MKS variations in the thioredoxin domain. Furthermore, TXNDC15 with mutations of two cysteine residues within the thioredoxin domain failed to rescue defects in TXNDC15-knockout cells, suggesting that TXNDC15 controls the TZ integrity from outside the TZ via its thioredoxin domain.

Keywords:  Cilia; Ciliopathy; Meckel syndrome; Thioredoxin; Transition zone

DOI:  https://doi.org/10.1242/jcs.262123
Immunity. 2024 Dec 12. pii: S1074-7613(24)00532-6. [Epub ahead of print]

The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance.

Yinfeng Xu, Qian Wang, Jun Wang, Chuying Qian, Yusha Wang, Sheng Lu, Lijiang Song, Zhengfu He, Wei Liu, Wei Wan.

  Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.

Keywords:  STING; TFEB; cGAS; innate immunity; lysosome

DOI:  https://doi.org/10.1016/j.immuni.2024.11.017
Sci Adv. 2024 Dec 20. 10(51): eadq4324

Deep representation learning of protein-protein interaction networks for enhanced pattern discovery.

Rui Yan, Md Tauhidul Islam, Lei Xing.

Protein-protein interaction (PPI) networks, where nodes represent proteins and edges depict myriad interactions among them, are fundamental to understanding the dynamics within biological systems. Despite their pivotal role in modern biology, reliably discerning patterns from these intertwined networks remains a substantial challenge. The essence of the challenge lies in holistically characterizing the relationships of each node with others in the network and effectively using this information for accurate pattern discovery. In this work, we introduce a self-supervised network embedding framework termed discriminative network embedding (DNE). Unlike conventional methods that primarily focus on direct or limited-order node proximity, DNE characterizes a node both locally and globally by harnessing the contrast between representations from neighboring and distant nodes. Our experimental results demonstrate DNE's superior performance over existing techniques across various critical network analyses, including PPI inference and the identification of protein functional modules. DNE emerges as a robust strategy for node representation in PPI networks, offering promising avenues for diverse biomedical applications.

DOI: https://doi.org/10.1126/sciadv.adq4324
Nat Commun. 2024 Dec 17. 15(1): 10670

Macronucleophagy maintains cell viability under nitrogen starvation by modulating micronucleophagy.

Ziyang Li, Keisuke Mochida, Hitoshi Nakatogawa.

Lysosome/vacuole-mediated intracellular degradation pathways, collectively known as autophagy, play crucial roles in the maintenance and regulation of various cellular functions. However, little is known about the relationship between different modes of autophagy. In the budding yeast Saccharomyces cerevisiae, nitrogen starvation triggers both macronucleophagy and micronucleophagy, in which nuclear components are degraded via macroautophagy and microautophagy, respectively. We previously revealed that Atg39-mediated macronucleophagy is important for cell survival under nitrogen starvation; however, the underlying mechanism remains unknown. Here, we reveal that defective Atg39-mediated macronucleophagy leads to the hyperactivation of micronucleophagy, resulting in the excessive transport of various nuclear components into the vacuole. Micronucleophagy occurs at the nucleus-vacuole junction (NVJ). We show that nuclear membrane proteins localized to the NVJ, including Nvj1, which is responsible for micronucleophagy, are degraded via macronucleophagy. Therefore, defective Atg39-mediated macronucleophagy results in the accumulation of Nvj1, which contributes to micronucleophagy enhancement. Blocking micronucleophagy almost completely suppresses cell death caused by the absence of Atg39, whereas enhanced micronucleophagy correlates with death in Atg39-mutant cells under nitrogen starvation. These results suggest that macronucleophagy modulates micronucleophagy in order to prevent the excess removal of nuclear components, thereby maintaining nuclear and cellular homeostasis during nitrogen starvation.

DOI: https://doi.org/10.1038/s41467-024-55045-9
MicroPubl Biol. 2024 ;2024

Proteomics analysis reveals the differential impact of the p97 inhibitor CB-5083 on protein levels in various cellular compartments of the HL-60 cell line.

Wenxuan Huang, Yanping Qiu, Diana Huynh, Ting-Yu Wang, Tsui-Fen Chou.

Human p97/VCP is a vital AAA ATPase (ATPase associated with diverse cellular activity) that plays critical roles in protein homeostasis by regulating autophagy, endosomal trafficking, and the ubiquitin-proteasome system. Global proteomics analysis of p97/VCP inhibition with CB-5083 has been performed in HCT116 colon cells. Here, we examined the impact of CB-5083 treatment in another cancer model, the HL-60 acute myeloid leukemia cell line, employing subcellular fractionation combined with label-free proteomics to analyze changes in protein levels across cytoplasmic, nuclear, and insoluble membrane protein compartments. The results reveal distinct compartment-specific protein regulation, providing insight into p97/VCP's cellular mechanisms and its potential for targeted therapeutic applications.

DOI: https://doi.org/10.17912/micropub.biology.001372
J Clin Invest. 2024 Dec 16. pii: e180347. [Epub ahead of print]134(24):

Disrupted uromodulin trafficking is rescued by targeting TMED cargo receptors.

Silvana Bazua-Valenti, Matthew R Brown, Jason Zavras, Magdalena Riedl Khursigara, Elizabeth Grinkevich, Eriene-Heidi Sidhom, Keith H Keller, Matthew Racette, Moran Dvela-Levitt, Catarina Quintanova, Hasan Demirci, Sebastian Sewerin, Alissa C Goss, John Lin, Hyery Yoo, Alvaro S Vaca Jacome, Malvina Papanastasiou, Namrata Udeshi, Steven A Carr, Nina Himmerkus, Markus Bleich, Kerim Mutig, Sebastian Bachmann, Jan Halbritter, Stanislav Kmoch, Martina Živná, Kendrah Kidd, Anthony J Bleyer, Astrid Weins, Seth L Alper, Jillian L Shaw, Maria Kost-Alimova, Juan Lorenzo B Pablo, Anna Greka.

  The trafficking dynamics of uromodulin (UMOD), the most abundant protein in human urine, play a critical role in the pathogenesis of kidney disease. Monoallelic mutations in the UMOD gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD-UMOD), an incurable genetic disorder that leads to kidney failure. The disease is caused by the intracellular entrapment of mutant UMOD in kidney epithelial cells, but the precise mechanisms mediating disrupted UMOD trafficking remain elusive. Here, we report that transmembrane Emp24 protein transport domain-containing (TMED) cargo receptors TMED2, TMED9, and TMED10 bind UMOD and regulate its trafficking along the secretory pathway. Pharmacological targeting of TMEDs in cells, in human kidney organoids derived from patients with ADTKD-UMOD, and in mutant-UMOD-knockin mice reduced intracellular accumulation of mutant UMOD and restored trafficking and localization of UMOD to the apical plasma membrane. In vivo, the TMED-targeted small molecule also mitigated ER stress and markers of kidney damage and fibrosis. Our work reveals TMED-targeting small molecules as a promising therapeutic strategy for kidney proteinopathies.

Keywords:  Genetic diseases; Nephrology; Protein misfolding; Protein traffic

DOI:  https://doi.org/10.1172/JCI180347
STAR Protoc. 2024 Dec 12. pii: S2666-1667(24)00674-9. [Epub ahead of print]5(4): 103509

Protocol to study secretome interactions using extracellular proximity labeling.

Joshua A Rich, Sadeechya Gurung, Sasha Coates-Park, Yueqin Liu, Anshika Govil, William G Stetler-Stevenson, David Peeney.

  Biotin ligase-based proximity ligation is a widely used, highly effective technique for the study of in vivo protein-protein interactions. However, there are few reports and little consensus on the most effective methods for studying the proximal interactomes of secreted factors. Here, we present a protocol for studying extracellular proximal interactomes using an adaptation of TurboID/BioID2-based proximity ligation. We describe steps for cell preparation, sample collection, and initial processing. We then detail procedures for biotinylated protein enrichment, on-bead digestion, and post-pull-down processing. For complete details on the use and execution of this protocol, please refer to Peeney et al.1.

Keywords:  Biotechnology and bioengineering; Cell Biology; Mass Spectrometry; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2024.103509
bioRxiv. 2024 Dec 05. pii: 2024.12.04.626665. [Epub ahead of print]

Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice.

Isabelle C Becker, Maria N Barrachina, Joshua Lykins, Virginia Camacho, Andrew P Stone, Bernadette A Chua, Robert A J Signer, Kellie R Machlus, Sidney W Whiteheart, Harvey G Roweth, Joseph E Italiano.

Megakaryocytes (MKs) are large, polyploid cells that contribute to bone marrow homeostasis through the secretion of cytokines such as transforming growth factor β1 (TGFβ1). During neoplastic transformation, immature MKs accumulate in the bone marrow where they induce fibrotic remodeling ultimately resulting in myelofibrosis. Current treatment strategies aim to prevent MK hyperproliferation, however, little is understood about the potential of targeting dysregulated cytokine secretion from neoplastic MKs as a novel therapeutic avenue. Unconventional secretion of TGFβ1 as well as interleukin 1β (IL1β) via secretory autophagy occurs in cells other than MKs, which prompted us to investigate whether similar mechanisms are utilized by MKs. Here, we identified that TGFβ1 strongly co-localized with the autophagy marker light chain 3B in native MKs. Disrupting secretory autophagy by inhibiting the small GTPase RhoA or its downstream effector Rho kinase (ROCK) markedly reduced TGFβ1 and IL1β secretion in vitro . In vivo , conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system limited megakaryocytosis and aberrant cytokine secretion in an MPL W515L -driven transplant model. Similarly, mice with a selective deletion of Rhoa from the MK and platelet lineage were protected from progressive fibrosis. Finally, disease hallmarks in MPL W515L -transplanted mice were attenuated upon treatment with the autophagy inhibitor hydroxychloroquine or the ROCK inhibitor Y27632, either as monotherapy or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data indicate that aberrant cytokine secretion is dependent on secretory autophagy downstream of RhoA, targeting of which represents a novel therapeutic avenue in the treatment of myelofibrosis.
One Sentence Summary: TGFβ1 is released from megakaryocytes via RhoA-mediated secretory autophagy, and targeting this process can alleviate fibrosis progression in a preclinical mouse model of myelofibrosis.

DOI: https://doi.org/10.1101/2024.12.04.626665
Mol Cancer. 2024 Dec 19. 23(1): 274

ZDHHC20 mediated S-palmitoylation of fatty acid synthase (FASN) promotes hepatocarcinogenesis.

Yaqi Mo, Yamei Han, Yang Chen, Chunling Fu, Qing Li, Zhuang Liu, Mingming Xiao, Bo Xu.

   BACKGROUND: Protein palmitoylation is a reversible fatty acyl modification that undertakes important functions in multiple physiological processes. Dysregulated palmitoylations are frequently associated with the formation of cancer. How palmitoyltransferases for S-palmitoylation are involved in the occurrence and development of hepatocellular carcinoma (HCC) is largely unknown.
METHODS: Chemical carcinogen diethylnitrosamine (DEN)-induced and DEN combined CCl4 HCC models were used in the zinc finger DHHC-type palmitoyltransferase 20 (ZDHHC20) knockout mice to investigate the role of ZDHHC20 in HCC tumourigenesis. Palmitoylation liquid chromatography-mass spectrometry analysis, acyl-biotin exchange assay, co-immunoprecipitation, ubiquitination assays, protein half-life assays and immunofluorescence microscopy were conducted to explore the downstream regulators and corresponding mechanisms of ZDHHC20 in HCC.
RESULTS: Knocking out of ZDHHC20 significantly reduced hepatocarcinogenesis induced by chemical agents in the two HCC mouse models in vivo. 97 proteins with 123 cysteine sites were found to be palmitoylated in a ZDHHC20-dependent manner. Among these, fatty acid synthase (FASN) was palmitoylated at cysteines 1471 and 1881 by ZDHHC20. The genetic knockout or pharmacological inhibition of ZDHHC20, as well as the mutation of the critical cysteine sites of FASN (C1471S/C1881S) accelerated the degradation of FASN. Furthermore, ZDHHC20-mediated FASN palmitoylation competed against the ubiquitin-proteasome pathway via the E3 ubiquitin ligase complex SNX8-TRIM28.
CONCLUSIONS: Our findings demonstrate the critical role of ZDHHC20 in promoting hepatocarcinogenesis, and a mechanism underlying a mutual restricting mode for protein palmitoylation and ubiquitination modifications.

Keywords:  FASN; Hepatocarcinogenesis; S-palmitoylation; ZDHHC20

DOI:  https://doi.org/10.1186/s12943-024-02195-5