pFind Studio: a computational solution for mass spectrometry-based proteomics
2024
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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COMMUNICATIONS BIOLOGY2024. Chen, Rui et al.
State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:Recruitment of non-canonical BCOR-PRC1.1 to non-methylated CpG islands via KDM2B plays a fundamental role in transcription control during developmental processes and cancer progression. However, the mechanism is still largely unknown on how this recruitment is regulated. Here, we unveiled the importance of the Poly-D/E regions within the linker of BCOR for its binding to KDM2B. Interestingly, we also demonstrated that these negatively charged Poly-D/E regions on BCOR play autoinhibitory roles in liquid-liquid phase separation (LLPS) of BCORANK-linker-PUFD/PCGF1RAWUL. Through neutralizing negative charges of these Poly-D/E regions, Ca2+ not only weakens the interaction between BCOR/PCGF1 and KDM2B, but also promotes co-condensation of the enzymatic core of BCOR-PRC1.1 with KDM2B into liquid-like droplet. Accordingly, we propose that Ca2+ could modulate the compartmentation and recruitment of the enzymatic core of BCOR-PRC1.1 on KDM2B target loci. Thus, our finding advances the mechanistic understanding on how the tethering of BCOR-PRC1.1 enzymatic core to KDM2B is regulated.Mechanistic studies on KDM2B interaction with BCOR/PCGF1 sheds light on the role of calcium and liquid-liquid phase separation in KDM2B recruiting BCOR-PRC1.1 on chromatin.
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SCIENCE2024. Yang, Zhenlin et al.
California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.
ABSTRACT:The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module. EP400 interacts with the transformation/transcription domain-associated protein (TRRAP) subunit by using a footprint that overlaps with that of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
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Elife2024. Daniel Ballmer et al.
Univ Oxford, Dept Biochem, Oxford, England; Inst Cell Biol, Wellcome Ctr Cell Biol, Sch Biol Sci, Edinburgh, Scotland
ABSTRACT:The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.
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Proceedings of the National Academy of Sciences2024. MenghanMei et al.
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
ABSTRACT:The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
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Food Hydrocolloids2024. YunkeYang et al.
College of Food Science, Southwest University, Chongqing, 400715, PR China
ABSTRACT:This work aimed to investigate the covalent crosslinking sites induced by transglutaminase (TGase, EC 2.3.2.13) in gelatin hydrogel and their impact on the gelatin molecular conformation. LC-MS/MS results demonstrated that Lys644 and Gln1351 of alpha 1 chain and Gln19 of alpha 2 chain were the main sites. The results of molecular dynamic (MD) simulation indicated that at the moderate degree of covalent crosslinking (13.55%), Radius of gyration (Rg) values decreased from 11.5 to 10.0 nm at 4 degrees C and led to a more compact structure, therefore enhancing structural tightness and gel strength. As the degree of crosslinking rose, the number of crosslinking sites gradually increased, with a predominant distribution in the C-terminal pro-peptide of gelatin chain. At the high crosslinking degree (37.48%), Rg value decreased at 100 degrees C and gelatin chains were connected through a "head to tail" linkage configuration, resulting in a significant reduction in the triple helix-like structure. The structure maintained the gel-forming ability at high temperature while lowering the aggregation energy of water molecules from -2.359 x 105 to -2.455 x 105 kJ/mol, leading to thermal irreversibility of gelatin hydrogel.
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