The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

Cell Communication and Signaling, Год журнала: 2023, Номер 21(1)

Опубликована: Сен. 30, 2023

Abstract Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation signal transduction, making it crucial to explore these understand complex biological processes. Traditional methods such as affinity purification yeast two-hybrid assays have been shown limitations, they can only isolate high-affinity molecular interactions under nonphysiological conditions or in vitro. Moreover, shortcomings for organelle isolation protein subcellular localization. To address issues, proximity labeling techniques developed. This technology not overcomes limitations traditional but also offers unique advantages studying spatial characteristics within living cells. Currently, this technique is indispensable research on mammalian nucleoprotein provides a reliable approach nonmammalian cells, plants, parasites viruses. Given advantages, article detailed introduction principles development enzymes. The focus summarizing recent applications TurboID miniTurbo mammals, microorganisms.

Язык: Английский

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Characterization of the intracellular neurexin interactome by in vivo proximity ligation suggests its involvement in presynaptic actin assembly

PLoS Biology, Год журнала: 2024, Номер 22(1), С. e3002466 - e3002466

Опубликована: Янв. 22, 2024

Neurexins are highly spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream neurexin’s largely invariant intracellular domain (ICD). Caenorhabditis elegans contains a single neurexin gene we have previously shown required for presynaptic assembly and stabilization. To gain insight into mediating functions, employed proximity ligation method, endogenously tagging with promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down mass spectrometry. We compared our experimental strain control in which neurexin, tagged was dispersed from active zones deletion its C-terminal PDZ-binding motif. Selection this strain, differs only synaptic localization, critical identifying interactions specifically occurring at synapses. Using approach, identified both novel interactors including zone scaffolds, actin-binding (including almost every member Arp2/3 complex), molecules, mediators RNA trafficking, protein synthesis degradation, among others. Characterization mutants candidate revealed they recapitulate aspects nrx-1(-) mutant phenotype, suggesting may be involved signaling. Finally, investigate possible role local actin assembly, depolymerizing sequestering peptides (DeActs) found led defects assembly. Together, these results suggest actin-assembly, furthermore highlight approach achieving high specificity vivo proteomics experiments.

Язык: Английский

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Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification–Mass Spectrometry

Journal of Proteome Research, Год журнала: 2023, Номер 22(10), С. 3383 - 3391

Опубликована: Сен. 15, 2023

We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation beads with Sulfo-NHS-Acetate leads chemical acetylation lysine residues, making ligands insusceptible Lys-C-mediated proteolysis. In contrast similar approaches, our procedure offers the advantage exclusively using nontoxic chemicals employing mild reaction conditions. After binding bait proteins treated beads, we employ a two-step digestion protocol sequential use Lys-C protease for on-bead followed by in-solution released trypsin. The implementation this results strong reduction contaminating ligand peptides, which allows significantly higher amounts sample be subjected LC-MS analysis, improving sensitivity quantitative accuracy.

Язык: Английский

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A new class of natural anthelmintics targeting lipid metabolism

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Янв. 2, 2025

Parasitic helminths are a major global health threat, infecting nearly one-fifth of the human population and causing significant losses in livestock crops. Resistance to few anthelmintic drugs is increasing. Here, we report set avocado fatty alcohols/acetates (AFAs) that exhibit nematocidal activity against four veterinary parasitic nematode species: Brugia pahangi, Teladorsagia circumcincta Heligmosomoides polygyrus, as well multidrug resistant strain (UGA) Haemonchus contortus. AFA shows efficacy H. polygyrus infected mice. In C. elegans, exposure affects all developmental stages, paralysis, impaired mitochondrial respiration, increased reactive oxygen species production damage. embryos, AFAs penetrate eggshell induce rapid arrest. Genetic biochemical tests reveal inhibit POD-2, encoding an acetyl CoA carboxylase, rate-limiting enzyme lipid biosynthesis. These results uncover new class affecting metabolism. Avocado effective nematodes, including drug strains, show safety mammalian cells. target metabolism, offering potential for treatment pathways.

Язык: Английский

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Proximity proteomics reveals unique and shared pathological features between multiple system atrophy and Parkinson’s disease

Acta Neuropathologica Communications, Год журнала: 2025, Номер 13(1)

Опубликована: Март 23, 2025

Abstract Synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are neurodegenerative diseases shared clinical pathological features. Aggregates of alpha-synuclein (αsyn) phosphorylated at serine 129 (PSER129) hallmarks synucleinopathies, which, for PD/DLB, found predominantly in neurons, whereas MSA, aggregates primarily oligodendroglia. It remains unclear whether the distinct presentations PD/DLB MSA manifestations unique or processes. Using in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compared aggregated αsyn-interactomes (BAR-PSER129) total (BAR-MJFR1) between (n = 5) 10) forebrain midbrain structures. Comparison PD/DLB-enriched proteins revealed 79 PD/DLB-differentially abundant only three MSA-differentially (CBR1, CRYAB, GFAP). Pathway enrichment analysis that vesicle/SNARE-associated pathways dominated interactions, was strongly enriched metabolic/catabolic, iron, cellular oxidant detoxification pathways. A subnetwork cytosolic antioxidant enzymes called peroxiredoxins drove pathway MSA. network 26 proteins, including neuronal-specific (e.g., SYNGR3) HSPA8 core, DLB/PD. Extracellular exosome were universally regardless BAR target protein. In conclusion, synucleinopathies have divergent convergent αsyn-aggregate indicating pathogenic mechanisms. uniquely involves processes glial cells, while vesicular neurons dominate PD/DLB. Shared specifically SYNGR3, suggest neuronal axons origin both diseases. provide αsyn protein interaction maps two synucleinopathies.

Язык: Английский

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TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Апрель 24, 2025

Язык: Английский

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A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes inC. elegans

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 14, 2024

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for conserved

Язык: Английский

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A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes in C. elegans

PLoS Genetics, Год журнала: 2024, Номер 20(8), С. e1011219 - e1011219

Опубликована: Авг. 22, 2024

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for conserved C . elegans phosphatase PTPN-22, establishing its role nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, found that loss PTPN-22 activity suppressed molting defects caused by loss-of-function mutations NIMA-related kinases NEKL-2 (human NEK8/NEK9) NEKL-3 NEK6/NEK7), which act at interface membrane trafficking actin To better understand carried out proximity labeling studies to candidate interactors during development. this approach identified CDC42 guanine-nucleotide exchange factor DNBP-1 DNMBP) as an vivo partner PTPN-22. Consistent with interaction, also nekl -associated defects. Genetic analysis, co-localization studies, revealed several epidermal adhesion complexes, including hemidesmosomes, suggesting plays broad maintaining structural integrity tissues. Localization implicated connected nucleocytoplasmic transport mRNA regulation, particularly within germline, nearly one-third proteins known P granule components. Collectively, these highlight utility combined proteomic approaches identifying gene functions.

Язык: Английский

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Recent Advances in Mass Spectrometry-based Protein Interactome Studies

Molecular & Cellular Proteomics, Год журнала: 2024, Номер unknown, С. 100887 - 100887

Опубликована: Ноя. 1, 2024

Язык: Английский

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Comparing alpha-synuclein-interactomes between multiple systems atrophy and Parkinsons disease reveals unique and shared pathological features.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Сен. 21, 2024

Abstract Introduction Primary synucleinopathies, such as Parkinson’s disease (PD), Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are neurodegenerative disorders some shared clinical pathological features. Aggregates of alpha-synuclein (αsyn) phosphorylated at serine 129 (PSER129) the hallmark which for PD/DLB found predominantly in neurons (Neuronal cytoplasmic inclusions “NCIs”), but MSA, aggregates primarily oligodendroglia (Glial “GCIs”). It remains unclear if distinct presentation MSA manifestations or processes. We hypothesize that synucleinopathies share common molecular Methods Using in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compare aggregated αsyn-interactomes (BAR-PSER129) total (BAR-MJFR1) between (n=5) (n=10) forebrain midbrain structures. Results For BAR-PSER129 BAR-MJFR1 captures, αsyn was most significantly enriched protein MSA. In PD/DLB, identified 194 αsyn-aggregate-interacting proteins, while 245 interacting proteins. contrast, brain, only 38 175 proteins were each capture, respectively. When comparing a high overlap (59.5%) observed captured whereas less (14.4%) BAR-PSER129. Direct comparison revealed 79 PD/DLB-associated three MSA-associated (CBR1, CRYAB, GFAP). Pathway enrichment analysis interactions dominated vesicle/SNARE-associated pathways, contrast to strongly metabolic/catabolic, iron, cellular oxidant detoxification pathways. A subnetwork cytosolic antioxidant enzymes called peroxiredoxins drove pathways network 26 including neuronal-specific (e.g., SNYGR3) HSPA8 core, DLB/PD. Extracellular exosome universally regardless BAR target protein. Conclusion Synucleinopathies have divergent convergent αsyn-aggregate interactions, indicating unique pathogenic mechanisms. uniquely involves processes glial cells, vesicular dominate PD/DLB. Shared specifically SNYGR3 (i.e., neuronal protein), suggest axons origin both diseases. conclusion, provide interaction maps two synucleinopathies.

Язык: Английский

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