pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
Communications Biology2023. Hu, Jianjian et al.
Hubei Univ, Coll Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Wuhan 430074, Peoples R China
ABSTRACT:Structural and mutagenic analyses reveal the mechanistic basis of azurin-mediated p53 stabilization and tumor suppression, and several affinity-enhancing azurin mutants are designed.Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.
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Journal of Agricultural and Food Chemistry2023. Ying Zhang et al.
Nanchang Univ, Sino German Joint Res Inst, Nanchang 330047, Peoples R China; Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China
ABSTRACT:Giventhat roasting changes the structure and allergenicity ofpeanut allergens, the structural information of peanut allergens mustbe expounded to explain the alteration in their allergenicity. Thiswork focused on allergen aggregations (AAs) in roasted peanuts. IgErecognition capability was assessed via western blot analysis. Thedisulfide bond (DB) rearrangement and chemical modification in AAswere identified by combining mass spectroscopy and software tools,and structural changes induced by cross-links were displayed by moleculardynamics and PyMOL software. Results showed that AAs were stronglyrecognized by IgE and cross-linked mainly by DBs. The types of DBrearrangement in AAs included interprotein (98 peptide pairs), intraprotein(22 peptide pairs), and loop-linked (6 peptides) DBs. Among allergens,Ara h 2 and Ara h 6 presented the most cysteine residues to cross-linkfwith others or themselves. DB rearrangement involved IgE epitopesand induced structural changes. Ara h 1 and Ara h 3 were predominantlychemically modified. Moreover, chemical modification altered the localstructures of proteins, which may change the allergenic potentialof allergens.
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Nature2023. Lulu Guo et al.
Shanghai Jiao Tong Univ, Ctr Brain Sci, Shanghai Childrens Med Ctr, Sch Med, Shanghai, Peoples R China; Shandong Univ, Adv Med Res Inst, Cheeloo Coll Med, Meili Lake Translat Res Pk, Jinan, Peoples R China; Shandong Univ, Dept Gen Surg, Qilu Hosp, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Inst, Sch Med, Shanghai, Peoples R China; Shandong Univ, Dept Biochem & Mol Biol, Sch Med, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Res Ctr Brain Sci & Brain Inspired Intel, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Shanghai Key Lab Childrens Environm Hlth, Dept Anat & Physiol, Minist Educ,Sch Med,Xinhua Hosp, Shanghai, Peoples R China; Peking Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Key Lab Mol Cardiovasc Sci,Minist Educ, Beijing, Peoples R China
ABSTRACT:Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with beta-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.
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Communications Biology2023. Caixuan Liu et al.
Univ Chinese Acad Sci, Beijing 100049, Peoples R China; 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 eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin-with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.Cryo-EM and XL-MS analyses reveal the conformational landscape of TRiCmediated tubulin folding along TRiC's ATPase cycle and the interaction sites between tubulin and the closed TRiC chamber.
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Antioxidants2023. Xiangzhe Zhou et al.
Beijing Inst Technol, Sch Life Sci, Beijing 100081, Peoples R China; Beijing Inst Technol, Inst Engn Med, Sch Med Technol, Beijing 100081, Peoples R China
ABSTRACT:Excessive hydrogen peroxide causes oxidative stress in cells. The oxidation of two tyrosine residues in proteins can generate o,o '-dityrosine, a putative biomarker for protein oxidation, which plays critical roles in a variety of organisms. Thus far, few studies have investigated dityrosine crosslinking under endogenous or exogenous oxidative conditions at the proteome level, and its physiological function remains largely unknown. In this study, to investigate qualitative and quantitative dityrosine crosslinking, two mutant Escherichia coli strains and one mutant strain supplemented with H2O2 were used as models for endogenous and exogenous oxidative stress, respectively. By integrating high-resolution liquid chromatography-mass spectrometry and bioinformatic analysis, we created the largest dityrosine crosslinking dataset in E. coli to date, identifying 71 dityrosine crosslinks and 410 dityrosine loop links on 352 proteins. The dityrosine-linked proteins are mainly involved in taurine and hypotaurine metabolism, citrate cycle, glyoxylate, dicarboxylate metabolism, carbon metabolism, etc., suggesting that dityrosine crosslinking may play a critical role in regulating the metabolic pathways in response to oxidative stress. In conclusion, we have reported the most comprehensive dityrosine crosslinking in E. coli for the first time, which is of great significance in revealing its function in oxidative stress.
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Nature2023. Guan, Haipeng et al.
State Key Laboratory of Molecular Oncology, MOE Key Laboratory of Protein Sciences, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, School of Medicine, Tsinghua University, Beijing, China
ABSTRACT:Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1-4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5-7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.
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Cell Reports2023. Dongdong Wu et al.
Univ Shanghai Sci & Technol, Sch Hlth Sci & Engn, Shanghai 200093, Peoples R China; Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, CAS Key Lab Quantitat Engn Biol, Shenzhen 518055, Peoples R China; Fudan Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Shanghai Key Lab Bioact Small Mol,Shanghai Med Col, Shanghai 200032, Peoples R China; Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Chem Biol, Shanghai 200032, Peoples R China
ABSTRACT:The present study examines whether there is a mechanism beyond the current concept of post-translational modifications to regulate the function of a protein. A small gas molecule, hydrogen sulfide (H2S), was found to bind at active-site copper of Cu/Zn-SOD using a series of methods including radiolabeled binding assay, X-ray absorption near-edge structure (XANES), and crystallography. Such an H2S binding enhanced the elec-trostatic forces to guide the negatively charged substrate superoxide radicals to the catalytic copper ion, changed the geometry and energy of the frontier molecular orbitals of the active site, and subsequently facil-itated the transfer of an electron from the superoxide radical to the catalytic copper ion and the breakage of the copper-His61 bridge. The physiological relevance of such an H2S effect was also examined in both in vitro and in vivo models where the cardioprotective effects of H2S were dependent on Cu/Zn-SOD.
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Molecular Cell2023. JanSchuhmacher et al.
Max Planck Inst Mol Physiol, Dept Struct Biochem, D-44227 Dortmund, Germany; Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany
ABSTRACT:The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A com-bination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and dem-onstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
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ACS Chemical Biology2023. Shengrong Li et al.
Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, MOE Key Lab Tumor Mol Biol, Guangzhou 510632, Peoples R China; Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, Guangzhou 510632, Peoples R China
ABSTRACT:Chemicalcross-linking of proteins coupled with massspectrometryanalysis (CXMS) is a powerful method for the study of protein structureand protein-protein interactions (PPIs). However, the chemicalprobes used in the CXMS are limited to bidentate reactive warheads,and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS)and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonylynamide, was developed as a new zero-length cross-linker that canconnect high-abundance carboxyl residues (D/E) with lysine (K) toform amide bonds in the absence of any catalyst. Significant improvementin the cross-linking efficiency and specificity in comparison withtraditional EDC/NHS was achieved with model proteins, which includesinter- and intramolecular conjugations. The cross-linked structureswere validated by X-ray crystallography. Importantly, this couplingreagent can be successfully used to capture interacting proteins inthe whole proteome and can be a useful reagent for probing potentialprotein-protein interactions in situ.
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Nature Structural & Molecular Biology2023. Zhouyan Guo et al.
Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Peoples R China
ABSTRACT:SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 angstrom. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3. ARM1 is in the inhibited state with the active site of Rpd3 blocked, whereas ARM2 is in an open conformation with the active site of Rpd3 exposed to the exterior space. The observed asymmetric architecture of Rpd3L is different from those of available structures of other class I HDAC complexes. Our study reveals the organization mechanism of the SIN3-HDAC complex and provides insights into the interaction pattern by which it targets histone deacetylase to chromatin.Here, Zhouyan et al. present the structure of yeast SIN3-HDAC complex Rpd3L, revealing a distinctive organization mechanism compared with other class I HDAC complexes. The authors observe that two distinct arms hang on a T-shaped scaffold formed by two coiled-coil domains.
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