pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
2023. Xuyan Shi et al.
National Institute of Biological Sciences, Beijing, Beijing, P. R. China
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P. R. China
Research Unit of Pyroptosis and Immunity, Chinese Academy of Medical Sciences and National Institute of Biological Sciences, Beijing, Beijing, P. R. China
Changping Laboratory, Beijing, P. R. China
Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, P. R. China
New Cornerstone Science Laboratory, Shenzhen, P. R. China
ABSTRACT:
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Nature Structural & Molecular Biology2023. Chen, Ying et al.
Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen,
ABSTRACT:Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-ELOC subunit heterodimer. ELOA contains a 'latch' that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center. The latch is required for the elongation-stimulatory activity of Elongin, but not for Pol II binding, indicating that Elongin functions by allosterically regulating the conformational mobility of the polymerase active center. Elongin binding to Pol II is incompatible with association of the super elongation complex, PAF1 complex and RTF1, which also contain an elongation-stimulatory latch element.Using cryo-EM, here the authors structurally delineate the Elongin-RNA polymerase II holocomplex. They show that Elongin allosterically regulates the transcribing RNA polymerase II via a latch that affects its conformational mobility.
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The Plant Cell2023. Jian Liu et al.
Huazhong Agr Univ, Natl Ctr Plant Gene Res, Wuhan 430070, Peoples R China; Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan 430070, Peoples R China
ABSTRACT:In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including beta-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit beta-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.Phosphatase-inactive LIKE SEX FOUR 1 interacts with moonlighting malate dehydrogenase to form a binary complex acting as a scaffold to recruit beta-amylase to promote starch degradation.
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Nature Structural & Molecular Biology2023. Park, Si Hoon et al.
Department of Life Sciences, Korea University, Seoul, South Korea
ABSTRACT:Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.
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2023. Ezeri{\c{n}}a, Daria et al.
Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels B-1050, Belgium
ABSTRACT:
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BMC bioinformatics2023. Zhou, Chen et al.
Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Pleinlaan 2, Brussels B-1050, Belgium
ABSTRACT:BACKGROUND: Cross-linking mass spectrometry (XL-MS) is a powerful technique for detecting protein-protein interactions (PPIs) and modeling protein structures in a high-throughput manner. In XL-MS experiments, proteins are cross-linked by a chemical reagent (namely cross-linker), fragmented, and then fed into a tandem mass spectrum (MS/MS). Cross-linkers are either cleavable or non-cleavable, and each type requires distinct data analysis tools. However, both types of cross-linkers suffer from imbalanced fragmentation efficiency, resulting in a large number of unidentifiable spectra that hinder the discovery of PPIs and protein conformations. To address this challenge, researchers have sought to improve the sensitivity of XL-MS through invention of novel cross-linking reagents, optimization of sample preparation protocols, and development of data analysis algorithms. One promising approach to developing new data analysis methods is to apply a protein feedback mechanism in the analysis. It has significantly improved the sensitivity of analysis methods in the cleavable cross-linking data. The application of the protein feedback mechanism to the analysis of non-cleavable cross-linking data is expected to have an even greater impact because the majority of XL-MS experiments currently employs non-cleavable cross-linkers.RESULTS: In this study, we applied the protein feedback mechanism to the analysis of both non-cleavable and cleavable cross-linking data and observed a substantial improvement in cross-link spectrum matches (CSMs) compared to conventional methods. Furthermore, we developed a new software program, ECL 3.0, that integrates two algorithms and includes a user-friendly graphical interface to facilitate wider applications of this new program.CONCLUSIONS: ECL 3.0 source code is available at https://github.com/yuweichuan/ECL-PF.git . A quick tutorial is available at https://youtu.be/PpZgbi8V2xI .
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Analytical Chemistry2023. Yanhong Hao et al.
Suming Chen
ABSTRACT:Cross-linking mass spectrometry (XL-MS) is a powerful tool for examining protein structures and interactions. Nevertheless, analysis of low-abundance cross-linked peptides is often limited in the data-dependent acquisition (DDA) mode due to its semistochastic nature. To address this issue, we introduced a workflow called 4D-diaXLMS, representing the first-ever application of four-dimensional data-independent acquisition for proteome-wide cross-linking analysis. Cross-linking studies of the HeLa cell proteome were evaluated using the classical cross-linker disuccinimidyl suberate as an example. Compared with the DDA analysis, 4D-diaXLMS exhibited marked improvement in the identification coverage of cross-linked peptides, with a total increase of 36% in single-shot analysis across all 16 SCX fractions. This advantage was further amplified when reducing the fraction number to 8 and 4, resulting in 125 and 149% improvements, respectively. Using 4D-diaXLMS, up to 83% of the cross-linked peptides were repeatedly identified in three replicates, more than twice the 38% in the DDA mode. Furthermore, 4D-diaXLMS showed good performance in the quantitative analysis of yeast cross-linked peptides even in a 15-fold excess amount of HeLa cell matrix, with a low coefficient of variation and high quantitative accuracies in all concentrations. Overall, 4D-diaXLMS was proven to have high coverage, good reproducibility, and accurate quantification for in-depth XL-MS analysis in complex samples, demonstrating its immense potential for advances in the field.
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Elife2023. Lu{\v{c}}i{\'c}, Iva et al.
Institute of Biology, Cellular Biophysics, Humboldt Universitt zu Berlin
ABSTRACT:The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.
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Cell Research2023. Dong, Shuqi et al.
CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, GIBH-HKU GuangdongHong Kong Stem Cell and Regenerative Medicine Research Centre, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China
ABSTRACT:In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP. These structures suggest that Rpd3S utilizes a conserved Sin3 basic surface to navigate through the nucleosomal DNA, guided by its interactions with H3K36 methylation and the extra-nucleosomal DNA linkers, to target acetylated H3K9 and sample other histone tails. Furthermore, our structures illustrate that Rpd3S reconfigures the DNA linkers and acts in concert with Hho1 to engage the NCP, potentially unraveling how Rpd3S and Hho1 work in tandem for gene silencing.
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Molecular Cell2023. GaurikaGarg et al.
Max Planck Institute for Multidisciplinary Sciences, Department of Molecular Biology, Am Fassberg 11, 37077 Gttingen, Germany
ABSTRACT:Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is 22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to 29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).
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