pFind Studio: a computational solution for mass spectrometry-based proteomics
2024
Cell Discovery2024. Tan, Yangxia et al.
Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine, Rui-Jin Hospital, School of Medicine and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
ABSTRACT:
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Science Advances2024. Bayly-Jones, Charles et al.
Cancer Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
ABSTRACT:
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Journal of Proteomics2024. Swati, Banerjee et al.
nstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nmst 542/2, 160 00 Praha, Czechia
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Nature Communications2024. Hayes, Brooke K et al.
Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC, Australia
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Nature communications2024. Zhao, Lili et al.
CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, State Key Laboratory of Medical Proteomics, Dalian
ABSTRACT:Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.
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NATURE CELL BIOLOGY2024. Stankunas, Edvinas et al.
Max Perutz Labs, Vienna Biocenter Campus, University of Vienna and Medical University of Vienna, Vienna, Austria
ABSTRACT:The nuclear basket attaches to the nucleoplasmic side of the nuclear pore complex (NPC), coupling transcription to mRNA quality control and export. The basket expands the functional repertoire of a subset of NPCs in Saccharomyces cerevisiae by drawing a unique RNA/protein interactome. Yet, how the basket docks onto the NPC core remains unknown. By integrating AlphaFold-based interaction screens, electron microscopy and membrane-templated reconstitution, we uncovered a membrane-anchored tripartite junction between basket and NPC core. The basket subunit Nup60 harbours three adjacent short linear motifs, which connect Mlp1, a parallel homodimer consisting of coiled-coil segments interrupted by flexible hinges, and the Nup85 subunit of the Y-complex. We reconstituted the Y-complex center dot Nup60 center dot Mlp1 assembly on a synthetic membrane and validated the protein interfaces in vivo. Here we explain how a short linear motif-based protein junction can substantially reshape NPC structure and function, advancing our understanding of compositional and conformational NPC heterogeneity.Stankunas and K & ouml;hler define how the nucleoplasmic portion of the nuclear pore complex (NPC), the basket, docks onto the NPC core by integrating AlphaFold-based interaction screens, electron microscopy, and membrane-templated reconstitutions.
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY2024. Novikova, Irina V et al.
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
ABSTRACT:The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O-2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The beta-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE(3)F(3) cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.
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ANALYTICAL CHEMISTRY2024. He, Yu et al.
Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, China
ABSTRACT:Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples. Compared with the conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 mu g DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 mu g HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm(2) in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each mu g of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 mu g HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. About 96% of intraprotein cross-links were within the maximal distance restraints of 26 & Aring;, and 75% of the identified PPIs reported by the STRING database were with high confidence (scores >= 0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.
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NATURE CELL BIOLOGY2024. Zhou, Bo et al.
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China
ABSTRACT:Gasdermin (GSDM) family proteins, known as the executors of pyroptosis, undergo protease-mediated cleavage before inducing pyroptosis. We here discovered a form of pyroptosis mediated by full-length (FL) GSDME without proteolytic cleavage. Intense ultraviolet-C irradiation-triggered DNA damage activates nuclear PARP1, leading to extensive formation of poly(ADP-ribose) (PAR) polymers. These PAR polymers are released to the cytoplasm, where they activate PARP5 to facilitate GSDME PARylation, resulting in a conformational change in GSDME that relieves autoinhibition. Moreover, ultraviolet-C irradiation promotes cytochrome c-catalysed cardiolipin peroxidation to elevate lipid reactive oxygen species, which is then sensed by PARylated GSDME, leading to oxidative oligomerization and plasma membrane targeting of FL-GSDME for perforation, eventually inducing pyroptosis. Reagents that concurrently stimulate PARylation and oxidation of FL-GSDME, synergistically promoting pyroptotic cell death. Overall, the present findings elucidate an unreported mechanism underlying the cleavage-independent function of GSDME in executing cell death, further enriching the paradigms and understanding of FL-GSDME-mediated pyroptosis.Zhou, Jiang, Dai et al report that upon ultraviolet-C radiation, full-length GSDME can induce pyroptosis without cleavage, likely due to conformational change and oxidative oligomerization after increased PARylation and mitochondrial lipid ROS levels.
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MOLECULAR CELL2024. Zhao, Haiyan et al.
Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical School, Aurora, CO 80045, USA
ABSTRACT:The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and cross- linking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.
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