pFind Studio: a computational solution for mass spectrometry-based proteomics
2021
MBio2021. Wang, B et al.
Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA.
ABSTRACT:The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.
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Nature communications2021. Huang, Y et al.
Sun Yat Sen Univ Canc Ctr, Collaborat Innovat Ctr Canc Med, State Key Lab Oncol South China, Guangdong Key Lab Nasopharyngeal Carcinoma Diag &, Guangzhou, Peoples R China.
ABSTRACT:Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors. The fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression can be physiologically important and clinically relevant in patients with TNBC. Notably, the combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7H3 axis might be a promising strategy for improving anti-tumor immune responses in patients with TNBC. B7H3 is a transmembrane B7 family checkpoint molecule present on many cancer cells. Here the authors show that FUT8 mediates fucosylation of B7H3 to limit the immune response to triple-negative breast cancer as a potentially targeted mechanism of non-responsiveness to current checkpoint therapies.
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JOURNAL OF CELL BIOLOGY2021. Yang, YH et al.
Chinese Acad Sci, Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing, Peoples R China.
ABSTRACT:Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.
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Analytical Chemistry2021. Qiang, Jiali et al.
Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 100 Haike Road, Pudong, Shanghai 201210, China
ABSTRACT:A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)". CADI is able to significantly reduce sample complexity by depleting 90% of the linear peptides while keeping the disulfide-bonded peptides. Furthermore, all CADI data can be directly analyzed by widely used protein database search engines, such as Mascot and MaxQuant. Our data show that CADI is able to sensitively identify disulfide bonds in peptides and proteins. However, CADI has not yet achieved a satisfied in-depth coverage on complex mammalian cell lysates due to the limited enzymatic activity of carboxypeptidase Y and low occurrences of disulfide bonds in a proteome. Altogether, CADI is a useful method that can get disulfide-linked peptides enriched and analyzed with regular search engines. CADI holds great potentials to deepen the analysis of disulfide bond and other types of cross-linked peptides on the proteome scale.
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Science2021. Chen, XZ et al.
Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, State Key Lab Genet Engn,Shanghai Key Lab Radiat, Shanghai, Peoples R China.
ABSTRACT:The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PICMediator (76 polypeptides, similar to 4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.
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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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eLife2021. Mast, FD et al.
Seattle Childrens Res Inst, Ctr Global Infect Dis Res, Seattle, WA 98101 USA; Rockefeller Univ, Lab Cellular & Struct Biol, 1230 York Ave, New York, NY 10021 USA; Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, 1230 York Ave, New York, NY 10021 USA; Univ Washington, Dept Pediat, Seattle, WA 98195 USA; Univ Washington, Dept Biochem, Seattle, WA 98195 USA
ABSTRACT:The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.
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NATURE STRUCTURAL & MOLECULAR BIOLOGY2021. Valencia-Sanchez, MI et al.
New York Univ Grossman Sch Med, Skirball Inst Biomol Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT:The cryo-EM structure of DNA-assembled histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus, a nucleocytoplasmic large DNA virus, reveals that these proteins form viral nucleosomes with highly conserved features when compared to canonical eukaryotic nucleosomes. Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3. These viral histones form 'forced' heterodimers, and a heterotetramer of four such heterodimers assembles DNA to form structures virtually identical to canonical eukaryotic nucleosomes.
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ANGEWANDTE CHEMIE-INTERNATIONAL EDITION2021. Liang, LJ et al.
Tsinghua Univ, Tsinghua Peking Ctr Life Sci, Minist Educ,Dept Chem, Key Lab Bioorgan Phosphorus Chem & Chem Biol,Ctr, Beijing 100084, Peoples R China.
ABSTRACT:Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated trans-thiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.
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Angewandte Chemie International Edition2021. Stieger, CE et al.
Forsch Verbund Berlin EV FMP, Leibniz Forschungsinst Mol Pharmakol, Chem Biol Dept, Campus Berlin Buch,Robert Roessle Str 10, D-13125 Berlin, Germany.
ABSTRACT:Diethynyl phosphinates were developed as bisfunctional electrophiles for the site-selective modification of peptides, proteins and antibodies. One of their electron-deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra- or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery. Finally, this reagent class was used to generate functional homogeneously rebridged antibodies that remain specific for their target. Their modular synthesis, thiol selectivity and conjugate stability make diethynyl phosphinates ideal candidates for protein conjugation for biological and pharmaceutical applications.
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