pFind Studio: a computational solution for mass spectrometry-based proteomics
2021
Journal of Biological Chemistry2021. Adamus, K et al.
Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic, Australia.
ABSTRACT:The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGAsubunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear. To investigate, we assayed the chromatinmodifying functions of SAGA and SLIK, revealing identical kinetics on minimal substrates in vitro. We also examined the binding of SAGA and SLIK to TBP and concluded that interestingly, both protein complexes have similar affinity for TBP. Additionally, despite the loss of Spt8 and C-terminus of Spt7 in SLIK, TBPprebound to SLIKis not released in the presence of TATA-box DNA, just like TBP prebound to SAGA. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (K-D = 10-17 nM), and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of the Spt8 subunit in SLIK neither drive any major conformational differences in its structure comparedwith SAGA, nor significantly affect HAT, DUB, or DNA-binding activities in vitro.
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Protein Expression and Purification2021. Gong, YY et al.
Tsinghua Univ, Ctr Synthet & Systemat Biol, Sch Life Sci, MOE Key Lab Bioinformat, Beijing 100084, Peoples R China.
ABSTRACT:Development of new affinity tags is important for recombinant protein expression and purification. Based on our earlier work, we devised an affinity tag by addition of two cysteine residues onto the N- and C-termini of the Fc-III peptide and designated as the Fc-III-4C tag, in which four cysteine residues form two disulfide linkages. The binding affinity of Fc-III-4C tag to human IgG is measured as 2.28 nM (K-d) and is 100 times higher than that of the Fc-III tag to IgG. Fc-III-4C tagged carbonic anhydrase (CA) can be effectively purified with IgG-immobilized beads, and Fc-III-4C tag does not possess adverse effects on the structure and stability of CA. Furthermore, the Fc-III-4C tagged protein binds to multiple transition metal ions, which enhances activities of enzymes that use metal ions as co-factors. These results suggest that Fc-III-4C tag is a useful tool for expression and purification of recombinant proteins and enhances the activities of some fusion proteins that use Zn2+ or Cu2+ as cofactors.
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CCS Chemistry2021. Zhou, Y et al.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023
ABSTRACT:Photosystem II (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex on the thylakoid membrane, is a primary target of light-induced photodamage. However, the overall molecular details of the conformation and composition dynamics of PSII photodamage are still controversial. In this study, we investigated systematically the dynamic conformation, degradation, and oxidation processes of PSII photodamage by integrating chemical cross-linking and top-down proteomics strategies. The dynamic disassembly of the PSII complex, as well as the degradation fragments from both extrinsic and intrinsic protein PSII subunits, were feasibly probed during the loss of O2-evolving activity. Some long-term controversial issues, including the activity loss of PSII complex occurs before the detachment of the Mn4CaO5cluster, were clarified. Finally, a detailed route map of dynamic PSII photodamage was outlined at the molecular level for the first time, which markedly enhanced our understanding of the molecular mechanism of photodamage.
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Nature communications2021. Chen, JH et al.
Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol Natl Ctr Prot Sci,Shanghai, Shanghai 200031, Peoples R China
ABSTRACT:The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS. Our structures reveal a slight leaning of PA28 alpha beta towards the alpha 3-alpha 4 side of iCP, disturbing the allosteric network of the gatekeeper alpha 2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28 alpha beta is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28 alpha beta -iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response. The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). Cryo-EM structures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP, combined with cross-linking data, reveal the complex architecture and suggest a distinct immunoproteasome activation mechanism.
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JOURNAL OF PHYSICAL CHEMISTRY B2021. Amos, SBTA et al.
Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT:alpha-Synuclein (alpha S) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of alpha S to membranes is a likely first step in the molecular pathophysiology of PD. The alpha S molecule can adopt multiple conformations, being largely disordered in water, adopting a beta-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of alpha-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of aS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-beta component (NAC) region of aS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of aS starting from the structure of aS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound aS indicate incipient beta-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound alpha S via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting alpha S advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.
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Advanced Biology2021. Liu, C et al.
Beihang Univ, Sch Med & Engn, Beijing Adv Innovat Ctr Big Data Based Precis Med, Beijing 100191, Peoples R China; Beihang Univ, Key Lab Big Data Based Precis Med, Minist Ind & Informat Technol, Beijing 100191, Peoples R China; Zhejiang Univ, MOE Lab Biosyst Homeostasis & Protect, Hangzhou 310058, Peoples R China; Zhejiang Univ, Life Sci Inst, Hangzhou 310058, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA; Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA; Chinese Acad Agr Sci, Inst Anim Sci, Beijing 100193, Peoples R China
ABSTRACT:Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by approximate to 92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.
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JACS2021. Klaus, M et al.
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue Strasse 15, Frankfurt am Main 60438, Germany
ABSTRACT:Polyketide synthases (PKSs) are versatile CC bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domainswell characterized on their ownare poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide B synthase by small-angle X-ray scattering and cross-linking mass spectrometry with coarse-grained structural modeling. The results of this hybrid approach shed light on the solution structure of a cis-AT type PKS module as well as its inherent conformational dynamics. Supported by a directed evolution approach, we also find that acyl carrier protein (ACP)-mediated substrate shuttling appears to be steered by a nonspecific electrostatic interaction network.
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY2021. Zhang, H et al.
Peking Univ, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Beijing 100871, Peoples R China.
ABSTRACT:The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells. By conjugation with a cellpenetrating peptide and a lysosomal-sorting sequence, the resulting GlueTAC chimera triggered the internalization and degradation of programmed death-ligand 1 (PD-L1), which provides a new avenue to target and degrade cell-surface proteins.
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Food Chemistry2021. Yang, N et al.
Northeast Agr Univ, Coll Food Sci & Technol, Key Lab Dairy Sci, Minist Educ, Harbin 150030, Heilongjiang, Peoples R China.
ABSTRACT:Polymer chemistry, rheology and cytotoxicity of cysteine initiated S-S redistribution in cold-extruded whey protein (TWPI) molecules were investigated. The locations of disulfide bonds in whey protein isolate (WPI), WPI dried without being extruded (OWPI) and cold-extruded WPI (TWPI), Cysteine (Cys)-treated WPI (WPI-Cys), OWPI (OWPI-Cys) and TWPI (TWPI-Cys) were precisely analyzed using liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software approaches. The numbers of intermolecular disulfide cross-linked peptides identified in Cys-treated samples increased by 4, 6 and 1, respectively, in the order of TWPI-Cys, OWPI-Cys and WPI-Cys. Fourier Transform infrared spectroscopy (FTIR) showed cysteine treatment loosed secondary structure of protein samples. Meanwhile, size exclusion chromatography (SEC) assay demonstrated the extensive polymerization in TWPI-Cys. Furthermore, Cys-treatment decreased the gelling temperature of TWPI to 57 degrees;C sharply. Cys-treated TWPI has 19.11 times storage modulus (G') and 25.86 times loss modulus (G") of Cys-untreated TWPI at 85 degrees C. Additionally, cell viability with Cys addition indicate modified whey proteins are not toxic to human umbilical vein endothelial cells (HUVECs).
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Journal of Proteome Research2021. Pirklbauer, GJ et al.
Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria.
ABSTRACT:Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct. We here present our new search engine, MS Annika, which can identify cross-linked peptides in MS2 spectra from a wide variety of cleavable cross-linkers. We show that MS Annika provides realistic estimates of FDRs without the need of arbitrary score cutoffs, being able to provide on average 44% more identifications at a similar or better true FDR than comparable tools. In addition, MS Annika can be used on proteome-wide studies due to fast, parallelized processing and provides a way to visualize the identified cross-links in protein 3D structures.
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