Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Shvarev, Dmitry; Schoppe, Jannis; König, Caroline; Perz, Angela; Füllbrunn, Nadia; Kiontke, Stephan; Langemeyer, Lars; Januliene, Dovile; Schnelle, Kilian; Kümmel, Daniel; Fröhlich, Florian; Moeller, Arne; Ungermann, Christian

doi:10.7554/elife.80901

Cited by 44 publications

(44 citation statements)

References 88 publications

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“…We previously proposed that HOPS may engage directly with ZF5.3 to promote escape during vesicle fusion; it may also promote trafficking into intraluminal vesicles as a prerequisite to endosomal escape . The observation that the high nuclear delivery efficiency of ZF5.3- t MeCP2 depends on both HOPS and CORVET, favors the former explanation and deserves further study, especially as HOPS and CORVET share 4 of 6 subunits in common. , Identification of those molecular features that promote productive interaction with HOPS and/or CORVET could improve the efficiency of other delivery strategies, including lipid nanoparticles, whose efficiency remains <10% …”

Section: Resultsmentioning

confidence: 97%

“…A class C core vacuole/ endosome tethering (CORVET) complex facilitates the fusion of Rab5 positive early endosomes, while the fusion of Rab7 positive late endosomes to lysosomes requires the homotypic fusion and protein sorting (HOPS)-tethering complex. 60,61 Previous mechanistic studies indicate that efficient cytosolic and nuclear trafficking of ZF5.3 relies on the HOPS complex, but not the analogous CORVET complex. 15 We thus sought to investigate if this dependence also held for the ZF5.3 conjugate of tMeCP2.…”

Section: Design Purification and Characterization Of Mecp2mentioning

confidence: 99%

“…15 The observation that the high nuclear delivery efficiency of ZF5.3-tMeCP2 depends on both HOPS and CORVET, favors the former explanation and deserves further study, especially as HOPS and CORVET share 4 of 6 subunits in common. 60,61 Identification of those molecular features that promote productive interaction with HOPS and/or CORVET could improve the efficiency of other delivery strategies, including lipid nanoparticles, whose efficiency remains <10%. 64 Delivered ZF-tMeCP2 Interacts with Partner Proteins in the NCoR/SMRT Complex.…”

Section: Design Purification and Characterization Of Mecp2mentioning

confidence: 99%

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Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2

et al. 2023

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The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-tMeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13–71, 313–484), binds DNA in a methylation-dependent manner in vitro, and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-tMeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.

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Section: Resultsmentioning

confidence: 97%

Section: Design Purification and Characterization Of Mecp2mentioning

confidence: 99%

Section: Design Purification and Characterization Of Mecp2mentioning

confidence: 99%

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Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2

et al. 2023

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“…Although CATCHR and HOPS/CORVET-family MTCs were long thought to be conformationally variable, single-particle cryo-EM studies of exocyst 11 , HOPS 62 , and now Dsl1:Qb:Qc have revealed relatively rigid cores with widely separated membrane-binding sites. It may instead be the attachments formed between MTCs and membranes that are flexible.…”

Section: Discussionmentioning

confidence: 99%

Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs

DAmico

Stanton

Shirkey

et al. 2023

Preprint

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Most membrane fusion reactions in eukaryotic cells are mediated by membrane tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here, we present a cryo-EM structure of the simplest known MTC, the 255-kDa Dsl1 complex, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with remarkable and unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

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“…By contrast, the C-terminal CNH domain has a β-propeller structure that binds to the small GTPase Rap2 [ 2 ]. Recently, the structures of the CNH domain, which has the homologous function of binding small GTPases, were elucidated [ 36 , 37 ]. The CNH domain of TNIK is separated from the kinase domain and subsequent helices by an approximately 500-residue unstructured region.…”

Section: Structural Model Of Full-length Tnik Indicates Scaffold Func...mentioning

confidence: 99%

Structural Insight into TNIK Inhibition

Kukimoto-Niino

Shirouzu²,

Yamada³

2022

IJMS

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TRAF2- and NCK-interacting kinase (TNIK) has emerged as a promising therapeutic target for colorectal cancer because of its essential role in regulating the Wnt/β-catenin signaling pathway. Colorectal cancers contain many mutations in the Wnt/β-catenin signaling pathway genes upstream of TNIK, such as the adenomatous polyposis coli (APC) tumor suppressor gene. TNIK is a regulatory component of the transcriptional complex composed of β-catenin and T-cell factor 4 (TCF4). Inhibition of TNIK is expected to block the aberrant Wnt/β-catenin signaling caused by colorectal cancer mutations. Here we present structural insights into TNIK inhibitors targeting the ATP-binding site. We will discuss the effects of the binding of different chemical scaffolds of nanomolar inhibitors on the structure and function of TNIK.

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Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Cited by 44 publications

References 88 publications

Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2

Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2

Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs

Structural Insight into TNIK Inhibition

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