bims-proteo 2022-05-01 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022‒05‒01
24 papers selected by
Eric Chevet
INSERM

Misfolded GBA/β-glucocerebrosidase impairs ER-quality control by chaperone-mediated autophagy in Parkinson disease.
Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response.
Emerging roles of endoplasmic reticulum proteostasis in brain development.
The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.
Pharmacological activation of ATF6 remodels the proteostasis network to rescue pathogenic GABAA receptors.
COPII vesicles in plant autophagy and endomembrane trafficking.
Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.
Sensor dimer disruption as a new mode of action to block the IRE1-mediated unfolded protein response.
Mitochondrial quality control in health and in Parkinson's disease.
The endoplasmic reticulum adopts two distinct tubule forms.
Deficiency in coatomer complex I causes aberrant activation of STING signalling.
Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs.
Acetylation of SCFD1 regulates SNARE complex formation and autophagosome-lysosome fusion.
Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.
Roadmap for Optimizing and Broadening Antibody-Based PROTACs for Degradation of Cell Surface Proteins.
USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.
Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis.
Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction.
Liquid phase separation of NEMO induced by polyubiquitin chains activates NF-κB.
Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.
Intrinsic role of chaperone-mediated autophagy in cancer stem cell maintenance.
The TORC1 phosphoproteome in C. elegans reveals roles in transcription and autophagy.
Evolutionary balance between foldability and functionality of a glucose transporter.
CROP: a retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system.

Autophagy. 2022 Apr 28.

Misfolded GBA/β-glucocerebrosidase impairs ER-quality control by chaperone-mediated autophagy in Parkinson disease.

Sheng-Han Kuo, Inmaculada Tasset, Ana Maria Cuervo, David Sulzer.

  Inhibition of chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, may contribute to pathogenesis in neurodegenerative diseases including Parkinson disease (PD). Pathogenic variants of PD-related proteins that reside in the cytosol, including SNCA/alpha-synuclein, LRRK2 (leucine rich repeat kinase 2), UCHL1 (ubiquitin Cterminal hydrolase 1) and VPS35 (VPS35 retromer complex component), exert inhibitory effects on CMA. Decreased CMA activity has also been reported in sporadic PD patients, consistent with an association between CMA inhibition and PD. We have now reported the first example of CMA dysfunction caused by a non-cytosolic PD-related protein, GBA/β-glucocerebrosidase, the most common genetic risk factor for PD, which uncovers a new role for CMA in endoplasmic reticulum (ER) quality control.

Keywords:  Chaperones; ER quality control; lysosomal enzymes; lysosomes; neurodegeneration; protein aggregation; protein trafficking; proteotoxicity

DOI:  https://doi.org/10.1080/15548627.2022.2071383
EMBO J. 2022 Apr 25. e109985

Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response.

Aleksandra P Pitera, Maria Szaruga, Sew-Yeu Peak-Chew, Steven W Wingett, Anne Bertolotti.

  Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.

Keywords:  GCN2; integrated stress response; stress responses; tRNA synthetase; translation

DOI:  https://doi.org/10.15252/embj.2021109985
Cells Dev. 2022 Apr 25. pii: S2667-2901(22)00017-1. [Epub ahead of print] 203781

Emerging roles of endoplasmic reticulum proteostasis in brain development.

Giselle Espinosa Vásquez, Danilo B Medinas, Hery Urra, Claudio Hetz.

  The development of the central nervous system requires a series of morphogenetic events that shape brain and spinal cord structures. Several brain regions and neural circuits are formed by differential gene expression patterns and cell migration events involving neurons. During neurogenesis and neuritogenesis, increased demand for protein synthesis occurs to express key neuronal proteins to generate axons, dendrites, and active synapsis. The endoplasmic reticulum (ER) is a central hub controlling protein homeostasis (proteostasis), impacting a wide range of cellular processes required for brain function. Although most of the field has focused on ER stress in neurodegenerative diseases marked by abnormal protein aggregation, accumulating evidence indicates that ER proteostasis contributes to brain development impacting processes such as neuronal migration, differentiation, and function. Here, we review emerging evidence linking neurodevelopment with ER proteostasis and its relevance to human disorders.

Keywords:  Brain development; Chaperones; Endoplasmic reticulum; Neurodevelopmental disorders; Proteostasis; Unfolded protein response

DOI:  https://doi.org/10.1016/j.cdev.2022.203781
Biol Rev Camb Philos Soc. 2022 Apr 26.

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Liliana M Almeida, Brígida R Pinho, Michael R Duchen, Jorge M A Oliveira.

  Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Keywords:  PERK; dynamics; endoplasmic reticulum; metabolic diseases; metabolism; mitochondria; neurodegeneration; stress; unfolded protein response

DOI:  https://doi.org/10.1111/brv.12860
Cell Biosci. 2022 Apr 27. 12(1): 48

Pharmacological activation of ATF6 remodels the proteostasis network to rescue pathogenic GABAA receptors.

Meng Wang, Edmund Cotter, Ya-Juan Wang, Xu Fu, Angela L Whittsette, Joseph W Lynch, R Luke Wiseman, Jeffery W Kelly, Angelo Keramidas, Ting-Wei Mu.

BACKGROUND: Genetic variants in the subunits of the gamma-aminobutyric acid type A (GABAA) receptors are implicated in the onset of multiple pathologic conditions including genetic epilepsy. Previous work showed that pathogenic GABAA subunits promote misfolding and inefficient assembly of the GABAA receptors, limiting receptor expression and activity at the plasma membrane. However, GABAA receptors containing variant subunits can retain activity, indicating that enhancing the folding, assembly, and trafficking of these variant receptors offers a potential opportunity to mitigate pathology associated with genetic epilepsy.RESULTS: Here, we demonstrate that pharmacologically enhancing endoplasmic reticulum (ER) proteostasis using small molecule activators of the ATF6 (Activating Transcription Factor 6) signaling arm of the unfolded protein response (UPR) increases the assembly, trafficking, and surface expression of variant GABAA receptors. These improvements are attributed to ATF6-dependent remodeling of the ER proteostasis environment, which increases protein levels of pro-folding ER proteostasis factors including the ER chaperone BiP (Immunoglobulin Binding Protein) and trafficking receptors, such as LMAN1 (Lectin Mannose-Binding 1) and enhances their interactions with GABAA receptors. Importantly, we further show that pharmacologic ATF6 activators increase the activity of GABAA receptors at the cell surface, revealing the potential for this strategy to restore receptor activity to levels that could mitigate disease pathogenesis.
CONCLUSIONS: These results indicate that pharmacologic ATF6 activators offer an opportunity to restore GABAA receptor activity in diseases including genetic epilepsy and point to the potential for similar pharmacologic enhancement of ER proteostasis to improve trafficking of other disease-associated variant ion channels implicated in etiologically-diverse diseases.

DOI: https://doi.org/10.1186/s13578-022-00783-w
FEBS Lett. 2022 Apr 29.

COPII vesicles in plant autophagy and endomembrane trafficking.

Baiying Li, Yonglun Zeng, Liwen Jiang.

  In eukaryotes, the endomembrane system allows for spatiotemporal compartmentation of complicated cellular processes. The plant endomembrane system consists of the endoplasmic reticulum (ER), the Golgi apparatus (GA), the trans-Golgi network (TGN), the multivesicular body (MVB), and the vacuole. Anterograde traffic from the ER to GA is mediated by coat protein complex II (COPII) vesicles. Autophagy, an evolutionarily conserved catabolic process that turns over cellular materials upon nutrient deprivation or in adverse environments, exploits double-membrane autophagosomes to recycle unwanted constituents in the lysosome/vacuole. Accumulating evidence reveals novel functions of plant COPII vesicles in autophagy and their regulation by abiotic stresses. Here, we summarize current knowledge about plant COPII vesicles in the endomembrane trafficking and then highlight recent findings showing their distinct roles in modulating the autophagic flux and stress responses.

Keywords:  COPII vesicle; autophagy; endomembrane trafficking; stress response

DOI:  https://doi.org/10.1002/1873-3468.14362
J Biol Chem. 2022 Apr 22. pii: S0021-9258(22)00416-1. [Epub ahead of print] 101976

Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.

Alexander Kröning, Johannes van den Boom, Matthias Kracht, Anja F Kueck, Hemmo Meyer.

  The protease SPRTN degrades DNA protein-crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability, and for designing strategies that target p97 in combination cancer therapy.

Keywords:  AAA+ ATPase; Cdc48; DNA-protein crosslink; SPRTN; VCP; Wss1; p97; protein unfolding

DOI:  https://doi.org/10.1016/j.jbc.2022.101976
Comput Struct Biotechnol J. 2022 ;20 1584-1592

Sensor dimer disruption as a new mode of action to block the IRE1-mediated unfolded protein response.

Kosala N Amarasinghe, Diana Pelizzari-Raymundo, Antonio Carlesso, Eric Chevet, Leif A Eriksson, Sayyed Jalil Mahdizadeh.

  The unfolded protein response (UPR) is activated to cope with an accumulation of improperly folded proteins in the Endoplasmic reticulum (ER). The Inositol requiring enzyme 1α (IRE1α) is the most evolutionary conserved transducer of the UPR. Activated IRE1 forms 'back-to-back'-dimers that enables the unconventional splicing of X-box Binding Protein 1 (XBP1) mRNA. The spliced XBP1 (XBP1s) mRNA is translated into a transcription factor controlling the expression of UPR target genes. Herein, we report a detailed in silico screening specifically targeting for the first time the dimer interface at the IRE1 RNase region. Using the database of FDA approved drugs, we identified four compounds (neomycin, pemetrexed, quercitrin and rutin) that were able to bind to and distort IRE1 RNase cavity. The activity of the compounds on IRE1 phosphorylation was evaluated in HEK293T cells and on IRE1 RNase activity using an in vitro fluorescence assay. These analyzes revealed sub-micromolar IC50 values. The current study reveals a new and unique mode of action to target and block the IRE1-mediated UPR signaling, whereby we may avoid problems associated with selectivity occurring when targeting the IRE1 kinase pocket as well as the inherent reactivity of covalent inhibitors targeting the RNase pocket.

Keywords:  Dimer disruptor; FDA approved drugs; IRE1α; MD simulations; Peptide docking; UPR

DOI:  https://doi.org/10.1016/j.csbj.2022.03.029
Physiol Rev. 2022 Apr 25.

Mitochondrial quality control in health and in Parkinson's disease.

Mohamed A Eldeeb, Rhalena A Thomas, Mohamed A Ragheb, Armaan Fallahi, Edward A Fon.

  As a central hub for cellular metabolism and intracellular signalling, the mitochondrion is a pivotal organelle, dysfunction of which has been linked to several human diseases including neurodegenerative disorders, and in particular Parkinson's disease. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses which increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to monitor, identify, repair and/or eliminate abnormal or misfolded proteins within the mitochondrion and/or the dysfunctional mitochondrion itself. Chaperones identify unstable or otherwise abnormal conformations in mitochondrial proteins and can promote their refolding to recover their correct conformation and stability. However, if repair is not possible, the abnormal protein is selectively degraded to prevent potentially damaging interactions with other proteins or its oligomerization into toxic multimeric complexes. The autophagic-lysosomal system and the ubiquitin-proteasome system mediate the selective and targeted degradation of such abnormal or misfolded protein species. Mitophagy (a specific kind of autophagy) mediates the selective elimination of dysfunctional mitochondria, in order to prevent the deleterious effects the dysfunctional organelles within the cell. Despite our increasing understanding of the molecular responses toward dysfunctional mitochondria, many key aspects remain relatively poorly understood. Herein, we review the emerging mechanisms of mitochondrial quality control including quality control strategies coupled to mitochondrial import mechanisms. In addition, we review the molecular mechanisms regulating mitophagy with an emphasis on the regulation of PINK1/PARKIN-mediated mitophagy in cellular physiology and in the context of Parkinson's disease cell biology.

Keywords:  PINK1/Parkin; Parkinson's disease; mitochondrial quality control; mitophagy; protein quality control

DOI:  https://doi.org/10.1152/physrev.00041.2021
Proc Natl Acad Sci U S A. 2022 May 03. 119(18): e2117559119

The endoplasmic reticulum adopts two distinct tubule forms.

Bowen Wang, Zhiheng Zhao, Michael Xiong, Rui Yan, Ke Xu.

  SignificanceThe endoplasmic reticulum (ER) is one of the most structurally visible and functionally important organelles in the cell. Utilizing superresolution microscopy, we here unveil that in the mammalian cell, the peripheral ER adopts two distinct, well-defined tubule forms of contrasting structures, molecular signatures, and functions, with one of the two curiously being ribbon-like, ultranarrow sheets of fixed widths. With fast multicolor microscopy, we further show how the two tubule forms dynamically interconvert while differentially accommodating proteins in the living cell.

Keywords:  ER tubules; ER-shaping proteins; endoplasmic reticulum; organelle morphology; superresolution microscopy

DOI:  https://doi.org/10.1073/pnas.2117559119
Nat Commun. 2022 Apr 28. 13(1): 2321

Deficiency in coatomer complex I causes aberrant activation of STING signalling.

Annemarie Steiner, Katja Hrovat-Schaale, Ignazia Prigione, Chien-Hsiung Yu, Pawat Laohamonthonkul, Cassandra R Harapas, Ronnie Ren Jie Low, Dominic De Nardo, Laura F Dagley, Michael J Mlodzianoski, Kelly L Rogers, Thomas Zillinger, Gunther Hartmann, Michael P Gantier, Marco Gattorno, Matthias Geyer, Stefano Volpi, Sophia Davidson, Seth L Masters.

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.

DOI: https://doi.org/10.1038/s41467-022-29946-6
Elife. 2022 Apr 25. pii: e76495. [Epub ahead of print]11

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs.

Antonio Cuevas-Navarro, Laura Rodriguez-Muñoz, Joaquim Grego-Bessa, Alice Cheng, Katherine A Rauen, Anatoly Urisman, Frank McCormick, Gerardo Jimenez, Pau Castel.

  RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

Keywords:  D. melanogaster; cancer biology; genetics; genomics; human; mouse

DOI:  https://doi.org/10.7554/eLife.76495
Autophagy. 2022 Apr 24. 1-15

Acetylation of SCFD1 regulates SNARE complex formation and autophagosome-lysosome fusion.

Hong Huang, Qinqin Ouyang, Kunrong Mei, Ting Liu, Qiming Sun, Wei Liu, Rong Liu.

  SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of acetylation (a post-translational modification) on macroautophagy/autophagy. Here, we demonstrate that both acetylation- and phosphorylation-dependent mechanisms control SCFD1's function in autophagosome-lysosome fusion. After detecting a decrease in the extent of SCFD1 acetylation under autophagy-stimulated conditions, we found that KAT2B/PCAF catalyzes the acetylation of residues K126 and K515 of SCFD1; we also showed that these two residues are deacetylated by SIRT4. Importantly, we showed that AMPK-controlled SCFD1 phosphorylation strongly disrupts the capacity of SCFD1 to interact with KAT2B, thus ensuring that the SCFD1 acetylation level remains low. Finally, we demonstrated that SCFD1 acetylation inhibits autophagic flux, specifically by blocking STX17-SNAP29-VAMP8 SNARE complex formation. Thus, our study reveals a mechanism through which phosphorylation and acetylation modifications of SCFD1 mediate SNARE complex formation to regulate autophagosome maturation.ACLY: ATP citrate lyase; CREB: cAMP responsive element binding protein; EBSS: nutrient-deprivation medium; EP300: E1A binding protein p300; KAT5/TIP60: lysine acetyltransferase 5; HOPS: homotypic fusion and protein sorting; MS: mass spectroscopy; SCFD1: sec1 family domain containing 1; SM: Sec1/Munc18; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UVRAG: UV radiation resistance associated.

Keywords:  Autophagosome; SCFD1; SNARE; autophagy; lysosome

DOI:  https://doi.org/10.1080/15548627.2022.2064624
Cell Rep. 2022 Apr 26. pii: S2211-1247(22)00497-1. [Epub ahead of print]39(4): 110736

Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.

Longlong Wang, Etori Aguiar Moreira, Georg Kempf, Yasuyuki Miyake, Blandina I Oliveira Esteves, Amal Fahmi, Jonas V Schaefer, Birgit Dreier, Yohei Yamauchi, Marco P Alves, Andreas Plückthun, Patrick Matthias.

  The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.

Keywords:  CP: Microbiology; DARPin; HDAC6; Zika virus; aggresome; influenza A virus; lysine deacetylases; stress granules; stress response; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2022.110736
ACS Chem Biol. 2022 Apr 28.

Roadmap for Optimizing and Broadening Antibody-Based PROTACs for Degradation of Cell Surface Proteins.

Josef A Gramespacher, Adam D Cotton, Paul W W Burroughs, Ian B Seiple, James A Wells.

Targeted protein degradation is a promising therapeutic strategy capable of overcoming the limitations of traditional occupancy-based inhibitors. By ablating all of the associated functions of a protein at once, the event-driven pharmacology of degrader technologies has recently enabled the targeting of proteins that have been historically deemed "undruggable". Most degradation strategies utilize the ubiquitin-proteasome system to mediate intracellular target degradation and are thus limited to targeting proteins with cytoplasmic domains. While some of these strategies, such as PROTACs, have shown great promise, there is a need for new modalities that can be applied to specifically target cell surface proteins. We previously described the development of an antibody-based PROTAC (AbTAC) that utilizes genetically encoded IgG bispecific antibody scaffolds to bring the cell surface E3-ligase RNF43 into the proximity of a membrane protein of interest (POI) to mediate its degradation. Here, we employ rational protein engineering strategies to interrogate and optimize the properties necessary for efficient degradation of two therapeutically important membrane proteins, PD-L1 and EGFR. We develop multiple antibodies to RNF43 and show that the specific antibody binding epitopes on RNF43 and the POI are more important than the affinities of the AbTAC antibodies. We further expand the available repertoire of E3 ligases by co-opting the E3-ligase ZNRF3 to degrade both PD-L1 and EGFR and show similar importance of epitope for degradation efficiency. Importantly, we show that both RNF43 and ZNRF3 AbTACs do not potentiate unwanted WNT signaling. Lastly, we find that these AbTACs can be even further improved by exploring various dual-binding and IgG scaffolds that range in flexibility, valency, and orientation of the binding arms. These structure-activity and mechanistic studies provide a roadmap for optimizing the development of AbTACs, thereby greatly expanding their utility for targeted cell surface protein degradation.

DOI: https://doi.org/10.1021/acschembio.2c00185
Nature. 2022 Apr 27.

USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.

Shuwen Zhang, Shitao Zou, Deyao Yin, Lihong Zhao, Daniel Finley, Zhaolong Wu, Youdong Mao.

Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels1-3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown4-7. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8-10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11-13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.

DOI: https://doi.org/10.1038/s41586-022-04671-8
Nat Cell Biol. 2022 Apr 25.

Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis.

Wei Yan, Minghui Cao, Xianhui Ruan, Li Jiang, Sylvia Lee, Adriana Lemanek, Majid Ghassemian, Donald P Pizzo, Yuhao Wan, Yueqing Qiao, Andrew R Chin, Erika Duggan, Dong Wang, John P Nolan, Jeffrey D Esko, Simon Schenk, Shizhen Emily Wang.

A decline in skeletal muscle mass and low muscular strength are prognostic factors in advanced human cancers. Here we found that breast cancer suppressed O-linked N-acetylglucosamine (O-GlcNAc) protein modification in muscle through extracellular-vesicle-encapsulated miR-122, which targets O-GlcNAc transferase (OGT). Mechanistically, O-GlcNAcylation of ryanodine receptor 1 (RYR1) competed with NEK10-mediated phosphorylation and increased K48-linked ubiquitination and proteasomal degradation; the miR-122-mediated decrease in OGT resulted in increased RYR1 abundance. We further found that muscular protein O-GlcNAcylation was regulated by hypoxia and lactate through HIF1A-dependent OGT promoter activation and was elevated after exercise. Suppressed O-GlcNAcylation in the setting of cancer, through increasing RYR1, led to higher cytosolic Ca2+ and calpain protease activation, which triggered cleavage of desmin filaments and myofibrillar destruction. This was associated with reduced skeletal muscle mass and contractility in tumour-bearing mice. Our findings link O-GlcNAcylation to muscular protein homoeostasis and contractility and reveal a mechanism of cancer-associated muscle dysregulation.

DOI: https://doi.org/10.1038/s41556-022-00893-0
Nat Commun. 2022 Apr 26. 13(1): 2242

Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction.

Sunan Li, Rongrong Li, Iqbal Ahmad, Xiaomeng Liu, Silas F Johnson, Liangliang Sun, Yong-Hui Zheng.

HIV-1 must counteract various host restrictions to establish productive infection. SERINC5 is a potent restriction factor that blocks HIV-1 entry from virions, but its activity is counteracted by Nef. The SERINC5 and Nef activities are both initiated from the plasma membrane, where SERINC5 is packaged into virions for viral inhibition or downregulated by Nef via lysosomal degradation. However, it is still unclear how SERINC5 is localized to and how its expression is regulated on the plasma membrane. We now report that Cullin 3-KLHL20, a trans-Golgi network (TGN)-localized E3 ubiquitin ligase, polyubiquitinates SERINC5 at lysine 130 via K33/K48-linked ubiquitination. The K33-linked polyubiquitination determines SERINC5 expression on the plasma membrane, and the K48-linked polyubiquitination contributes to SERINC5 downregulation from the cell surface. Our study reveals an important role of K130 polyubiquitination and K33/K48-linked ubiquitin chains in HIV-1 infection by regulating SERINC5 post-Golgi trafficking and degradation.

DOI: https://doi.org/10.1038/s41467-022-30026-y
Mol Cell. 2022 Apr 19. pii: S1097-2765(22)00293-3. [Epub ahead of print]

Liquid phase separation of NEMO induced by polyubiquitin chains activates NF-κB.

Mingjian Du, Chee-Kwee Ea, Yan Fang, Zhijian J Chen.

  The NF-κB essential modulator (NEMO) is a regulatory subunit of the IκB kinase (IKK) complex that phosphorylates the NF-κB inhibitors IκBs. NEMO mediates IKK activation by binding to polyubiquitin chains (polyUb). Here, we show that Lys63(K63)-linked or linear polyUb binding to NEMO robustly induced the formation of liquid-like droplets in which IKK was activated. This liquid phase separation of NEMO was driven by multivalent interactions between NEMO and polyUb. Both the NEMO ubiquitin-binding (NUB) domain and the zinc-finger (ZF) domain of NEMO mediated binding to polyUb and contributed to NEMO phase separation and IKK activation in cells. Moreover, NEMO mutations associated with human immunodeficiency impaired its phase separation. These results demonstrate that polyUb activates IKK and NF-κB signaling by promoting the phase separation of NEMO.

Keywords:  IKK; NEMO; NF-κB; inflammation; innate immunity; liquid phase separation; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2022.03.037
Proc Natl Acad Sci U S A. 2022 May 03. 119(18): e2118465119

Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.

Anwar Sadat, Satyam Tiwari, S Sunidhi, Aseem Chaphalkar, Manisha Kochar, Mudassar Ali, Zainab Zaidi, Akanksha Sharma, Kanika Verma, Kannan Boosi Narayana Rao, Manjul Tripathi, Asmita Ghosh, Deepika Gautam, Atul, Arjun Ray, Koyeli Mapa, Kausik Chakraborty.

  SignificanceHsp60/10 chaperonins are critical for cellular proteostasis in all kingdoms of life. In this study, we present that Hsp60/10 across different species have differences in the cavity properties and correlatively in their capability to remove entropic traps in folding pathways of GroEL/ES substrates; this is affected majorly by differences in the negative-charge density inside the chaperonin cavity. This dissimilarity leads to a remarkable difference between Hsp60/10 homologs in buffering mutational variations. However, most of them can remove nonnative contacts during folding of their substrates and alter the way the polypeptide chain undergoes hydrophobic collapse. We show that these homologs may have evolved specific modes of folding assistance by modulating cavity properties according to the requirements of their substrates.

Keywords:  chaperone; mutational buffering; protein folding

DOI:  https://doi.org/10.1073/pnas.2118465119
Autophagy. 2022 Apr 27. 1-2

Intrinsic role of chaperone-mediated autophagy in cancer stem cell maintenance.

Jaione Auzmendi-Iriarte, Ander Matheu.

  Chaperone-mediated autophagy (CMA) is a selective type of autophagy specialized in the individual degradation of targeted proteins. Its impact in any cancer stem cell (CSC) subtype remained elusive. In a recent study, we characterized the expression of LAMP2A and CMA activity in glioblastoma revealing its enrichment in a glioma stem cell (GSC) subpopulation. LAMP2A downregulation diminishes proliferation and self-renewal and induces apoptosis in GSCs in vitro, whereas it delays tumor progression in vivo. The underlying molecular signature of CMA comprises several proteomic and transcriptomic pathways with special relevance to mitochondrial function, the interferon pathway and extracellular matrix interactions. Remarkably, these activities are translated into the clinical scenario, as glioblastoma (GBM) samples show increased expression of LAMP2 compared to healthy tissue, with this expression being positively associated with malignancy grade, TMZ resistance and lower patient survival. These results reveal a novel function of CMA as an intrinsic regulator of GSC tumorigenic properties and highlight its relevance in GBM progression.

Keywords:  CMA; Cancer stem cell; LAMP2A; glioblastoma; proteomic and transcriptomic analysis

DOI:  https://doi.org/10.1080/15548627.2022.2069450
iScience. 2022 May 20. 25(5): 104186

The TORC1 phosphoproteome in C. elegans reveals roles in transcription and autophagy.

Aileen K Sewell, Zachary C Poss, Christopher C Ebmeier, Jeremy R Jacobsen, William M Old, Min Han.

  The protein kinase complex target of rapamycin complex 1 (TORC1) is a critical mediator of nutrient sensing that has been widely studied in cultured cells and yeast, yet our understanding of the regulatory activities of TORC1 in the context of a whole, multi-cellular organism is still very limited. Using Caenorhabditis elegans, we analyzed the DAF-15/Raptor-dependent phosphoproteome by quantitative mass spectrometry and characterized direct kinase targets by in vitro kinase assays. Here, we show new targets of TORC1 that indicate previously unknown regulation of transcription and autophagy. Our results further show that DAF-15/Raptor is differentially expressed during postembryonic development, suggesting a dynamic role for TORC1 signaling throughout the life span. This study provides a comprehensive view of the TORC1 phosphoproteome, reveals more than 100 DAF-15/Raptor-dependent phosphosites that reflect the complex function of TORC1 in a whole, multi-cellular organism, and serves as a rich resource to the field.

Keywords:  Cell biology; Developmental biology; Functional aspects of cell biology; Omics; Proteomics

DOI:  https://doi.org/10.1016/j.isci.2022.104186
Nat Chem Biol. 2022 Apr 28.

Evolutionary balance between foldability and functionality of a glucose transporter.

Hyun-Kyu Choi, Hyunook Kang, Chanwoo Lee, Hyun Gyu Kim, Ben P Phillips, Soohyung Park, Charlotte Tumescheit, Sang Ah Kim, Hansol Lee, Soung-Hun Roh, Heedeok Hong, Martin Steinegger, Wonpil Im, Elizabeth A Miller, Hee-Jung Choi, Tae-Young Yoon.

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3's microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

DOI: https://doi.org/10.1038/s41589-022-01002-w
EMBO J. 2022 Apr 25. e109646

CROP: a retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system.

Thibault Courtellemont, Maria Giovanna De Leo, Navin Gopaldass, Andreas Mayer.

  Endo-lysosomal compartments exchange proteins by fusing, fissioning, and through endosomal transport carriers. Thereby, they sort many plasma membrane receptors and transporters and control cellular signaling and metabolism. How the membrane fission events are catalyzed is poorly understood. Here, we identify the novel CROP complex as a factor acting at this step. CROP joins members of two protein families: the peripheral subunits of retromer, a coat forming endosomal transport carriers, and membrane inserting PROPPINs. Integration into CROP potentiates the membrane fission activity of the PROPPIN Atg18 on synthetic liposomes and confers strong preference for binding PI(3,5)P2 , a phosphoinositide required for membrane fission activity. Disrupting CROP blocks fragmentation of lysosome-like yeast vacuoles in vivo. CROP-deficient mammalian endosomes accumulate micrometer-long tubules and fail to export cargo, suggesting that carriers attempt to form but cannot separate from these organelles. PROPPINs compete for retromer binding with the SNX-BAR proteins, which recruit retromer to the membrane during the formation of endosomal carriers. Transition from retromer-SNX-BAR complexes to retromer-PROPPIN complexes might hence switch retromer activities from cargo capture to membrane fission.

Keywords:  autophagy; endosomes; lysosomes; retromer; yeast

DOI:  https://doi.org/10.15252/embj.2021109646